Method for rapidly determining effective sterilization, deimmunization, and/or disinfection

ABSTRACT

A method for rapidly determining effective sterilization, deimmunization, and/or disinfection of equipment and/or supplies by a device. The method includes providing a defined surrogate protein having a predetermined sequence representative of an infectious agent potentially contaminating the equipment and/or the supplies to be sterilized, deimmunized, and/or disinfected by the device. The defined surrogate protein having the predetermined sequence is subjected to sterilization, deimmunization, or disinfection. The effectiveness of the sterilization deimmunization, or disinfection is rapidly determined by determining if the defined surrogate protein has been destroyed.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/472,499 filed Mar. 29, 2017, and claims thebenefit of and priority thereto under 35 U.S.C. §§ 119, 120, 363, 365,and 37 C.F.R. § 1.55 and § 1.78, which is incorporated herein by thisreference, and U.S. patent application Ser. No. 15/472,499 claims thebenefit of and priority to U.S. Provisional Application Ser. No.62/314,617 filed Mar. 29, 2016, under 35 U.S.C. §§ 119,120,363, 365, and37 C.F.R. § 1.55 and § 1.78, which is incorporated herein by thisreference.

REFERENCE TO A SEQUENCE LISTING

The Sequence Listing, which is a part of the present disclosure,includes a text file comprising primer nucleotide and/or amino acidsequences of the present invention. The subject matter of the SequenceListing is incorporated herein by reference in its entirety. Theinformation recorded In computer readable form is identical to thewritten sequence listing.

FIELD OF THE INVENTION

This invention relates to a method for rapidly determining effectivesterilization, deimmunization, and/or disinfection.

BACKGROUND OF THE INVENTION

A wide range of infectious agents, including infectious proteins, sporeforming bacteria, vegetative bacteria, fungus and viruses have majorimpacts in medical settings. The process to remove infectious organismsor render them non-infectious from medical equipment makes use of a widerange of sterilization devices or equipment and disinfection devices andprocesses. The CDC lists examples of infectious agents andmicroorganisms by resistance to standard disinfection and sterilizationprocesses. See Table 1 below from CDC's Guideline for Disinfection andSterilization in Healthcare Facilities, 2008.

Some infectious agents, such as the HIV virus, may be easy to removefrom medical equipment. Many infectious agents, including vegetativebacteria, are moderately difficult to eliminate. Other infectiousagents, such as prions, can only be destroyed by extremely harshconditions that damage and/or destroy modern medical equipment. Failureto eliminate infectious agents from medical equipment before use can putpatients at extreme risk of injury and death.

TABLE 1 Decreasing order of resistance of infectious agents andmicroorganisms to disinfection and sterilization. Agent Category ExampleOrganisms or Diseases Prions Creutfeldt-Jakob Disease Bacterial sporesBacillius atrophaeus Coccidica Cryptosporidium Mycobacteria M.tuberculosis, M. terrae Nonlipid or small viruses polio, coxsackie FungiAspergillus, Candida Vegetative bacteria S. aureus, P. aeruginosa Lipidof medium-sized viruses HIV, herpes, hepatitis B

Some conventional methods to determine if sterilization equipmentfunctions effectively may rely on FDA approved Biologic Indicatorprocess (BI strips) in a multi-step process. This widely acceptedconventional process starts with filter papers infused with a definednumber of bacterial spores (BI strips). The BI strips are subjected to astandard cycle by the sterilization equipment or device, e.g., anethylene oxide (EtO) sterilization, radiation, or steam sterilizationequipment which is being qualified. After the sterilization process iscompleted, the treated strips are then placed in a defined bacterialmedia for growth, frequently for days to weeks. If no growth is seenafter the defined period, the sterilization process by the medicalequipment being certified is declared a success. Together thiscombination of supplies and techniques is the approved process toqualify sterilization equipment in positive or negative process. Ifthere is growth, the sterilization equipment fails and if there is nogrowth the sterilization equipment passes.

The conventional biologic indicator tests may use one of three differentspecies of bacteria. The standard species used to test the effectivenessof ethylene oxide (EtO) sterilization is B. atrophaeus. To test theeffectiveness of gamma radiation sterilization, the bacteria speciesused is B. pumilis. To test the effectiveness of steam sterilization thebacteria species used is G. stearothermophilus. However, the threespecies that are used to qualify sterilization capacity of equipment arenot bacteria that commonly cause disease in humans. Instead, the speciesare surrogate species, strains of soil bacteria that form highpersistent spores. They are used instead of medically relevantinfectious agents, because, inter alia, the spores of the bacteria areextremely difficult to damage such that they can no longer replicate,and if for some reason a health care worker or patient accidently comesin contact with the spores through use or on improperly cleanedequipment, there is very little chance that the human will become ill.As spores from the surrogate species are scientifically known to be moredifficult to destroy than medically relevant species, such as Polio orS. aureus, e.g., Methicillin-resistant Staphyloccus aureaus (MRSA), whenthe sterilization equipment is qualified to destroy all spores on a BItest strip, the FDA accepts that the equipment is also able to destroyall organisms that rank lower for resistance to sterilization.

The conventional methods discussed above used to qualify effectivesterilization, deimmunization, or disinfection may only measure theability of the surrogate organisms to grow after sterilizationtreatment. However, such conventional methods do not indicate how thesurrogate organisms are damaged and/or destroyed resulting in theabsence of growth. Conventional surrogate testing methods also requirethe accurate production, storage, transport and handling of 10 thousandto 100 million pure bacteria spores, proper control of growth medias,extended period of growth of the specific spores and careful protectionof all growth materials for environmental contamination to qualify ifall the test surrogate organisms were completely eliminated. If anycomponent of the process is not vigorously controlled, the sterilizationqualification could give false positive or false negative results. Falsepositive results will trigger extensive effort to unnecessarily repairsterilization equipment as well as the recall of days or weeks ofsterilized medical equipment and the patients treated with suchequipment. False negative results are worse because they will result indefective sterilization equipment being used and the resultingcontaminated medical equipment endangering patients.

For other infectious organisms, such as members of the bacterial generaClostridium, Staphylococcus or fungal genera Trichophyton or Candida,and the like, specific tests for each genera may be based on similarfundamentals. See Table 2 below for a list of common bacteria and fungusgenera and species having an impact on human medicine.

TABLE 2 Common bacteria and fungus genera and species having an impacton human medicine. Health Care Application - Problem pathogen orBL1/sterilization Genus Example Species testing organism. BacillusBacillus subtilis Research organism, used as an “indicator organism”during disinfection testing. BL1. Bacillus atrophaeus Used as an“indicator organism” during gas (EtO) sterilization procedure. BL1.Bacillus pumilis Used as an “indicator organism” during radiationsterilization procedure. BL1. Geobacillus stearothermophilus Used as an“indicator organism” (formerly B. stearothermophilus) during steamsterilization procedure. BL1. Bacillus anthracis Causes anthrax.Bacillus cereus Causes food poisoning similar to that caused byStaphylococcus. Clostridium Clostridium sporogenes Used as a surrogatefor C. botulinum when testing the efficacy of commercial sterilization.Clostridium tetani Causes tetanus. Clostridium botulinum Causes botulismpoisoning. Clostridium perfringens Causes gas gangrene Clostridiumdifficile Causes C. dif GI infection. Clostridium novyi Causes a widerange of human and animal infections depending on type. MycobacteriumMycobacterium tuberculosis Major cause of human tuberculosis.Mycobacterium africanum Slow growing form of tuberculosis. Mycobacteriumcaprae More rare form of human tuberculosis. Mycobacterium kansasiiChronic human pulmonary disease resembling tuberculosis (involvement ofthe upper lobe). Mycobacterium ulcerans Infects the skin andsubcutaneous tissues, giving rise to indolent nonulcerated and ulceratedlesions. Mycobacterium interjectum Chronic lymphadenitis Mycobacteriumleprae Causes leprosy Mycobacterium lepromatosis Causes leprosyMycobacterium terrae Causes serious skin infections that are relativelyresistant to antibiotic therapy. Used to study effectiveness ofdisinfection processes for reusable medical instruments. Mycobacteriumgastri Casual resident of human stomachs, but not considered anetiologic agent of disease. (BL1). Staphylococcus Staphylococcus aureusCauses a variety of infections in the body, including boils, cellulitis,abscesses, wound infections, toxic shock syndrome, pneumonia, and foodpoisoning. Substrain - Methicillin-resistant Staphylococcus aureus(MRSA). Sub-strain - Vancomycin-resistant Staphylococcus aureus (VRSA) -acquired gene from VRE. Staphylococcus capitis Associated withprosthetic valve endocarditis, forms biofilms. Staphylococcusepidermidis Hospital-acquired concern as it forms biofilms catheters orother surgical implants. Staphylococcus haemolyticus Second-mostfrequently isolated hospital-acquired Infection, often associated withthe insertion of medical devices; highly antibiotic- resistant phenotypeand able to form biofilms. Staphylococcus lugdunensis Wide variety ofinfections including cardiovascular infections, osteomyelitis andprosthetic/native joints infections, skin and soft-tissue infection,central nervous infections, peritonitis, endocephalitis, and urinarytract infections. Staphylococcus saccharolyticus May cause of infectiveendocarditis. Staphylococcus saprophyticus Common cause of community-acquired urinary tract infections. Staphylococcus auricularisOccasionally can be involved with human skin infections. SalmonellaSalmonella enterica Causes food poisoning. Enterococcus Enterococcusfaecalis Can cause endocarditis and septicemia, urinary tractinfections, meningitis, and other infections. Substrain -Vancomycin-resistant Enterococcus (VRE). Enterococcus faecium Neonatalmeningitis or endocarditis. Substrain - Vancomycin-resistantEnterococcus (VRE). Enterococcus gallinarum Known to cause outbreaks andspread in hospitals. Enterococcus hirae Endocarditis and septicemia inhumans. Enterococcus malodoratus Frequently the cause of hospital-acquired noscomial infections, bloodstream infections, and urinary tractinfections. Escherichia Escherichia coli Some serotypes can causeserious food poisoning in their hosts. Substrain K-12 strain commonlyused in recombinant DNA work (BL1). Substrain O157:H7 causes seriousillness or death in the elderly, the very young, or theimmunocompromised. Substrain O104:H4, can trigger major cause offoodborne illness and lead to hemolytic-uremic syndrome (HUS).Escherichia fergusonii Known to infect open wounds and may also causebacteraemia or urinary tract infections; highly resistant to theantibiotic ampicillin and some also resistant to gentamicin andchloramphenicol. Helicobacter Helicobacter pylori Cause gastritis andulcers. Helicobacter hepaticus May be associated with Crohn's diseaseand ulcerative colitis. Helicobacter bilis May be associated withCrohn's disease and ulcerative colitis. Helicobacter ganmani May beassociated with Crohn's disease and ulcerative colitis. KlebsiellaKlebsiella pneumoniae Causes pneumonia, urinary tract infections,septicemia, meningitis, diarrhea, and soft tissue infections; naturallyresistant to many antibiotics. Substrain - CREs - carbapenem- resistantKlebsiella pneumoniae (CRKP). Klebsiella oxytoca Cause colitis andsepsis. Neisseria Neisseria gonorrhoeae Causes Gonorrhea. Neisseriameningitidis Causes meningitis. Pseudomonas Pseudomonas aeruginosa Amultidrug resistant pathogen associated with hospital-acquiredinfections such as ventilator- associated pneumonia and various sepsissyndromes. Common in CF patients. Pseudomonas mendocina Occasionallycauses hospital- acquired infections, such as infective endocarditis andspondylodiscitis. Pseudomonas fluorescens Produces enzymes that causemilk to spoil and occasionally infects immunocompromised patients.Pseudomonas putida Used in bioremediation, or the use of microorganismsto biodegrade oil. Trichophyton Trichophyton rubrum Most common cause ofathlete's (Fungus) foot, fungal infection of nail, jock itch, andringworm. Trichophyton tonsurans Causes ringworm infection of the scalp.Trichophyton interdigitale One of three common fungi which causeringworm. Trichophyton mentagrophytes Causes tinea infections includingathlete's foot, ringworm, jock itch, and similar infections of the nail,beard, skin and scalp. Trichophyton concentricum Associated with theskin infection tinea imbricate. Candida Candida albicans Dimorphicfungus that grows both (Fungus) as yeast and filamentous cells;Responsible for 50-90% of all cases of candidiasis in human. Importantcauses of morbidity and mortality in immunocompromised patients.Biofilms may form on the surface of implantable medical devices. Causeof 85-95% of vaginal infections cases are responsible for physicianoffice visits every year. Candida dubliniensis A fungal opportunisticpathogen originally isolated from AIDS patients. It is also occasionallyisolated from immunocompetent individuals. Candida tropicalis Commonpathogen in neutropaenic hosts; research suggests that C. tropicalis,working synergistically with Escherichia coli and Serratia marcescens.May cause or contribute to Crohn's disease Candida auris Causescandidiasis in humans; often acquired in the hospital when human immunesystems are weakened; causes fungemia, yielding candidemia (systemiccandidiasis); attracted clinical attention because of multidrugresistance.

Examples of standardized methods for sterilization or disinfection (A)and standardized testing methods protocols (B) used to determine theeffectiveness of sterilization or disinfection, as shown in Table 3below:

TABLE 3 Approved Methods of Sterilization or Disinfection and QualifyingTest Protocols (A) Standard Methods for Preparing Healthcare EquipmentDisinfection Sterilization Alcohol Steam Sterilization Chlorine andChlorine Compounds Flash Sterilization Formaldehyde Ethylene Oxide “Gas”Sterilization Glutaraldehyde Hydrogen Peroxide Gas Plasma HydrogenPeroxide Peracetic Acid Sterilization Iodophors Ionizing RadiationOrtho-phthalaldehyde Dry-Heat Sterilizers Peracetic Acid LiquidChemicals Peracetic Acid and Hydrogen Peroxide Performic Acid PhenolicsFiltration Quaternary Ammonium Compounds Microwave Radiation Glass Bead“Sterilizer” Pasteurization Vaporized Hydrogen Peroxide Flushing- andWasher-Disinfectors Ozone Formaldehyde Gaseous Chlorine DioxideVaporized Peracetic Acid Infrared radiation (B) Standard Test toQualtify Healthcare Equipment Test Name Example Test Species BI(ethylene oxide (EtO) sterilization) B. atrophaeus BI (gamma radiationsterilization) B. pumilis BI (steam sterilization) G. stearothermophilusAOAC Sporicidal Efficacy Test Method Clostridium sporogenes Bacillussubtilis AOAC Tuberculosis Rate of Kill Mycobacterium terrae AOAC UseDilution Test Pseudomonas aeruginosa Staphylococcus aureus Salmonellaenterica AOAC Fungicidal Efficacy Test Method Trichophytonmentagrophytes

For each standard test protocol, a define number of organisms are placedon a carrier, such as tube, filter paper, or coated on and in a testsolid surface. The specific organisms may be a particular infectiousspecies or could be a surrogate species of the same genus that isclosely related to the infectious species. In all cases, the species,carrier and growth conditions are defined by the FDA and/or theAssociation of Analytical Communities (AOAC) protocol. Followingtreatment with sterilization or disinfection equipment, the carrier withthe specific species sample is placed into ideal growing conditions forthe particular test species. After a required period in culture, usually2 to 30 days, the culture is monitored. If no growth is observed, thesterilization or disinfection equipment is declared to be operatingwithin required parameters.

In addition to enabling growth and infectivity, protein components ofinfectious organisms could trigger severe immunogenic or allergicreactions in susceptible individuals even at very low level. Examplesinclude mold proteins that are able to trigger severe allergic reactionseven if the mold has been rendered no longer able to grow. Immunogenicproteins can also occur in food such as gliadin, a highly immunogenicprotein component of the seed storage protein gluten in wheat andrelated grains. Gliadin can trigger reactions in most individualssuffering from Crohn's disease. It is critical that immunogenic proteinsare completely removed from any equipment that will be used inconjunction with susceptible individuals.

A wide range of pathogenic organisms use a multicopper oxidase with 3cupredoxin superfamily domains for growth and survival. As disclosedherein, the loci suf I that contains a critical protein that confersdifferent functions depending on the genus (bacteria or fungus) and thiscritical protein can be targeted. Depending on the genus, the suf I lociencoded protein can have different names. The functions of the proteinencoded by suf I include cell division (FtsP), formation of spore coatproteins (CotA), chromosome partitioning, inorganic ion transport, andmetabolism and cell wall, membrane, and envelope formation. As theprotein product of the suf I loci are absolutely critical for thesurvival of the spores (in spore forming bacteria) and/or growth (allbacteria and fungus), if the protein product of the suf I loci isirreversibly fragmented into short polypeptides and amino acids, thebacteria or fungus cannot survive. Additionally, it is likely that asterilization method that clearly demonstrates fragmentation of theprotein product of the suf I loci would also fragment other proteins inthe bacterium or fungus. Bacteria and fungus can be divided intodistinct genus each containing multiple species. Many species also havesubspecies that carry unique characteristics include multi-drugresistance. In human health situations, certain bacteria and fungusspecies and subspecies are of major concern because they are capable ofcausing disease. Related species may be used in medical research, e.g.,E. coli KI2, or as indicator species for qualification of sterilization,e.g., B. atrophaeus used to qualify gas sterilization. See Table 2 abovefor a list of common bacteria and fungus genera and species with impacton human medicine.

Prions are a unique category of a transmissible infections agent thatcomprised only of protein, without DNA or RNA. Prions can cause a widerange of neurodegenerative diseases known as transmissible spongiformencephalopathies (TSE) or prion diseases including the new variantCreutzfeldt-Jakob disease (nvCJD). See Table 4 below. Infectious prionsare in fact an abnormally folded brain protein. This brain protein(Protease resistant Proteins, PrP) can be folded into two differentstructural (tertiary) forms, the normal brain protein, PrP_(c,) and theabnormal, disease triggering form, PrP_(sc.) The disease triggeringform, PrP_(sc,) is found in high quantity in the brain of infectedhumans and animals and can be transferred to a new host with thetransfer of infected material. Once in the new host, the abnormallyfolded protein (PrP_(sc)) causes disease symptoms by promoting theunfolding of the normal host protein (PrP_(c)) and refolding into thedisease causing form (PrP_(sc)). PrP proteins can also be partiallycleave and still retain their infectious characteristics. Full lengthmature PrP protein (both PrP_(c) and PrP_(sc)) is 209 amino acids long.Limited proteolysis of PrP_(sc) will cleave amino acids from the aminoterminus resulting in another infectious protein form PrP 27-30 that isapproximately 142 amino acids long. Additional cleavage thatsignificantly reduces the 142 amino acid long PrP 27-30 is needed torender the PrP protein irreversibly non-infectious. Although mostinfectious agents can be permanently rendered non-infectious by heat orsteam, these methods are not sufficient to eliminate infectious prionsfrom medical equipment.

TABLE 4 Example of Prion Diseases in Different Species and PotentialOrigin of the Infectious Protein. Disease Species Potential OriginDisease Species Potential Origin Creutzfeldt-Jakob Human InheritedScrapie Sheep and Inherited/ disease (CJD) Goat environmental NewVariant Human Consumption, Bovine Spongiform Cattle ConsumptionCreutzfeldt-Jakob Medical Encephalopathy disease (CJD) Contamination(BSE) Fatal Familial Human Inherited Transmissible Mink MinkEnvironmental Insomnia (FFI) Encephalopathy (TME) Gerstmann-StrausslerHuman Inherited Chronic Wasting Mule Deer Environmental disease (GSD)Disease (CWD) and Elk Huntington disease- Human Inherited FelineSpongiform Cats Consumption like type 1 (HDL1) Encephalopathy (FSE) KuruHuman Consumption of Exotic Ungulate Nyala and Environmental HumanBrains Encephalopathy Greater (EUE) Kudu

As discussed above, prions are abnormally folded protease resistantproteins (PrP_(sc)) that cause disease symptoms by promoting theunfolding of normal proteins (PrP_(c)) and refolding into the diseasecausing protein form (PrP_(sc)). As the level of the PrP_(sc) rises inthe patient's brain, symptoms of progressive dementia, myoclonicseizures, abnormalities of high cortical function, cerebellar andcorticospinal disturbances develop. The period between infection anddevelopment of disease can extend for years to decades. The duration ofdisease symptoms is variable but is typically 8 to 18 months.

Once prion proteins fold into the infectious form (PrP_(sc)), they areextremely difficult to render non-infectious. Conventional methods tosterilize medical equipment contaminated with prions, such as high heatto promote loss of function of other protein types by triggering loss oftertiary structure, are ineffective because unlike most proteins, thedenatured prion proteins, both infectious and non-infectious, willspontaneously refold by themselves back to their pre-treatment forms. Insome cases, conventional methods may actually result in refolded intoinfectious form promoting the conversion of the non-infection prionprotein into the infectious prion protein.

To render infectious proteins such as prions irreversiblynon-infectious, all infectious proteins must be fragmented into smallpolypeptides, amino acids or components. The only currently approvedconventional method for this process Is harsh treatment of medicalequipment and supplies with caustic soda, an extremely harsh processthat frequently damages and/or destroys medical equipment

Determining whether or not an infectious prion (PrP_(sc)) sample hasbeen permanently destroyed can be extremely difficult and timeconsuming. Conventional methods for determining whether an infectiousprion has been permanently destroyed require that after attempteddeactivation, the PrP_(sc) sample is injected into a matched susceptibleanimal that is then followed for an extended time to see if the animaldevelops disease. In larger animals, the process can take years, buteven in a small animal such as a mouse, the test can take months. Asthere is a potential for inter-animal variation and poor test accuracy,a large animal test pool is required to obtain relatively accurateresults.

Immunogens may include a wide range of molecules including proteins thatcan trigger dramatic immunologic responses in susceptible individuals.The responses can trigger serious allergic reactions on the skin (e.g.,poison ivy rash), in the gut (e.g., triggering a flare-up in Crohn'sdisease), in the lung (e.g., asthma) or a systemic response (e.g.,anaphylaxis). Protein immunogens are a special class of immunogensproduced by a wide range of bacteria, fungus (e.g. mold) or plants andcan be difficult to destroy. An example of a common plant immunogen isgluten. Common grains such as various strains of wheat, farro, rye andspelt are derived from wild and domesticated grains of the Triticum,Aegilops and Secale genera. Common to all these species is the seedstorage protein complex called gluten. When seeds are ground into flour,the gluten protein complex gives bread dough its elastic quality andbread its spongy texture. Unfortunately gluten is comprised of severalproteins including Gliadin (also called Prolamin) which triggers severeT cell attack on the gut of patients with the autoimmune disease Celiacdisease (CD). Gliadins can be typed as α, γ, and ω with a small proteaseresistant fragment (p57-73) of α-gliadins triggering the most severedestructive T cell response. As a results CD patients must not onlyavoid products containing gluten, but also need to be extremely carefulto avoid small amounts of residual α-gliadin that may contaminate foodpreparation utensils.

Protease resistant proteins like α-gliadin are resistant to destructionso it is critical that devices and methods used to destroy them andother immunogens (also called allergens) can be easily checked to ensurethey are operating at peak efficiency. If not, residual allergens cantrigger life threatening responses in sensitive patients. The process ofremoving immunogens by deimmunization methods or devices is calleddeimmunization. The ability to test for the destruction of differentimmunogens on surfaces is not standardized. Usually affected patientsare subjected to skin test regiments to determine their individualreactions to different candidate immunogens/allergens. The patient isthen advised to avoid all immunogen contact and discard any materialspotential contaminated with the specific immunogen or allergen. Incooking and manufacturing situations, extreme care must be taken toavoid potential cross contamination to the point that food packaginglabels frequently carry warning labels about the potential issues.

Thus, a method is needed to determine irreversible destruction ofproteins critical for the growth of infectious organisms, immunogenicproteins, and/or infectious proteins (e.g., prions) and thus rapidly andaccurately determines the effectiveness of sterilization,deimmunization, and/or disinfection of equipment or supplies by adevice. With such a method for rapidly determining effectivesterilization, deimmunization, and/or disinfection, medical personneland patients can have confidence that the medical equipment used forpatient treatment is not contaminated with potentially lethal orimmunogenic proteins. Without such a method, medical personnel maybelieve they are using properly sterilized equipment and then laterdiscover that they have accidentally exposed their patients to lethalinfections and harmful immune reactions

SUMMARY OF THE INVENTION

In one aspect, a method for rapidly determining effective sterilization,deimmunization, and/or disinfection of equipment and/or supplies by adevice is featured. The method includes providing a defined surrogateprotein having a predetermined sequence representative of an infectiousagent potentially contaminating the equipment and/or the supplies to besterilized, deimmunized, and/or disinfected by the device. The definedsurrogate protein having the predetermined sequence is subjected tosterilization, deimmunization, or disinfection. The effectiveness of thesterilization, deimmunization, or disinfection is rapidly determined bydetermining if the defined surrogate protein having the predeterminedsequence has been destroyed.

In one embodiment, the defined surrogate protein may include proteinscritical for stability, growth and/or infectious capacity of infectiousagents. The defined surrogate protein may include a protein critical forstability, growth and/or infectious capacity of surrogate organisms ofinfectious agents. The infectious agent may include one or more of: aninfectious protein, an infectious spore forming bacteria, an infectiousvegetative bacteria, an infections fungus, and an infectious virus. Thedefined surrogate protein may include pathogenic proteins, proteinscritical for the growth of infectious agents, and immunogenic proteins.The predetermined sequence may be defined by the sequence:

(SEQ ID NO: 1)         10         20         30         40MYNYTSAKYE VPIAIQDRSF NEDGSLNFPS EGDNPTIHPY         50WQPEFFGDTI MVNGRVWPNM NVDMTRYRFR LLNGSNARFYNLKFSNGMQF WQIGTDGGYL NKFVPLTSLL ISPGERADILVDFTEIPAGT RIILNNDANA PYPTGDAPDK DTTGQIMQFT VQHNDHHHHH H

The defined surrogate protein for SEQ ID NO: 1 may be at least 95%homologous the predetermined sequence or substantial fragment of thepredetermined sequence. The predetermined sequence may be defined by thesequence:

(SEQ ID NO: 2)         10         20         30         40MTLEKTYYEV TMEECTHQLH RDLPPTRLWG YNGLFPGPTI         50EVKRNENVYV KWMNNLPSTH FLPIDHTIHH SDSQHEEPEVKTVVHLHGGV TPDDSDGYPE AWFSKDFEQT GPYFKREVTHYPNQQRGAIL WYHDHAMALT RLNVYAGLVG AYIIHDPKEK RLKAHHHHH

The defined surrogate protein for SEQ ID NO: 2 may be at least 95%homologous to the predetermined sequence or substantial fragment of thepredetermined sequence. The predetermined sequence may be defined by thesequence:

(SEQ ID NO: 3)         10         20         30         40MTGMPEGEGV DSNLLGGDGG DIAIPYYLIN GRIPVAATSF         50KAKPGQRIRI RIINSAADTA FRIALAGHSM TVTHTDGYPVIPTEVDALLI GMAERYDVMV TAAGGVFPLV ALAEGKNALA RALLSTGAGS FPDHHHHHH

The defined surrogate protein for SEQ ID NO: 3 may be at least 95%homologous to the predetermined sequence or substantial fragment of thepredetermined sequence.

The predetermined sequence may be defined by the sequence:

(SEQ ID NO: 4)         10         20         30         40MTGYKNYTLK AQKGKTEFYK NNFSNTLGYN GNLLGPTLKL         50KKGDKVKIKL INNLDENTTF HWHGLEVNGK VDGGPSQVIKPGKEKTIKFE VNQDSATLWY HPHPSPNTAK QVYNGLSGLL YIEDSKKNHH HHHH

The defined surrogate protein SEQ ID NO: 4 may be at least 95%homologous to the predetermined sequence or substantial fragment of thepredetermined sequence.

The predetermined sequence may be defined by the sequence:

(SEQ ID NO: 5)         10         20         30         40MTGFRHEKVL CLKTWHVDEQ GAFTPFSVPR QAAREGTRGR         50YSTINGKHVP TIDLPAGQIV RVRLLNVDNT VTYRLNPNGEARIYAVDGHP VEPRGFEGQY WIGPGMRLEL ALKVPEAGTE LSLRDGPVRL ATIRSVAHHH HHH

The defined surrogate protein SEQ ID NO: 5 may be at least 95%homologous to the predetermined sequence or substantial fragment of thepredetermined sequence. The predetermined sequence may be defined by thesequence:

(SEQ ID NO: 6)         10         20         30         40MTITLEWSVT TGYRRLDGVK KRVYLINGLF PGPTIEARSG         50DSLQVQVTNN IQDEGLVIHW HGLHMRGANH MDGVTGVTQCPIVPGDSMLY NFTISQSQSG TFWYHAHSAL QRAEGLYGGF VVHKPSTHHH HHH

The defined surrogate protein SEQ ID NO: 6 may be at least 95%homologous to the predetermined sequence or substantial fragment of thepredetermined sequence. The predetermined sequence may be defined by thesequence:

(SEQ ID NO: 7)         10         20         30         40MTAETHTWYF KTSWVDANPD GVFPRKMIGF NDSWPLPTLR         50VKKGDTVNLY LINGFDDRNT SLHFHGLFQH GTNQMDGPEMVTQCPIPPGE TFLYNFTVDD QVGSYWYHSH TSGQYGDGMR GVFIIEDHHH HHH

The defined surrogate protein SEQ ID NO: 7 may be at least 95%homologous to the predetermined sequence or substantial fragment of thepredetermined sequence. The predetermined sequence may be defined by thesequence:

(SEQ ID NO: 8)         10         20         30         40MKTVRVPVPQ PQPQNPSQPQ PQRQVPLVQQ QQFPGQQQQF         50PPQQPYPQPQ PFPSQQPYLQ LQPFPQPQPF PPQLPYHHHH HH

The defined surrogate protein SEQ ID NO: 8 may be at least 95%homologous to the predetermined sequence or substantial fragment of thepredetermined sequence. The rapidly determining may include a sensitiveprotein analysis procedure. The sensitive protein analysis procedure mayinclude one or more of: a Western Blot analysis, a protein assayanalysis, a magnetic separation analysis, a peptide analysis, a massspectrometry analysis, and a gas chromatography analysis. The sensitiveprotein analysis procedure may include fluorescence analysis of proteinscovalently crosslinked on a solid surface. The sensitive proteinanalysis procedure may include fluorescence analysis of proteinscovalently crosslinked on magnetic beads. The defined surrogate proteinhaving the predetermined sequence may be disposed on a surface, disposedon a test strip, disposed in or on a vessel, on a tube, or in or on aholder. The holder may be disposed to receive a flow of a sterilizationagent, a deimmunization agent or a disinfection agent.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Other objects, features and advantages will occur to those skilled inthe art from the following description of a preferred embodiment and theaccompanying drawings, in which:

FIG. 1 is a schematic block diagram showing the primary steps of oneembodiment of the method for rapidly determining effectivesterilization, deimmunization, and/or disinfection of this invention;

FIG. 2 is a schematic diagram showing examples of multi-well glassslides having samples of one or more of the defined surrogate proteinhaving a predetermined sequence in wells which are subjected tosterilization, deimmunization, or disinfection to provide a visualdepiction of the effectiveness of sterilization, deimmunization, and/ordisinfection:

FIG. 3 is a schematic block diagram showing one example of themulti-well glass slides shown in FIG. 2 placed in a holder disposed atthe end of a flow of sterilization, deimmunization, or disinfectionagent in accordance with one embodiment of this invention;

FIG. 4 shows an example of an amino acid comparison of human PrPproteins with a selection of other species of PrP proteins;

FIG. 5 shows an example of a Western gel where the recombinant proteinruns approximately 28 kDa inside;

FIG. 6 shows an example of a Western Blot where the absence of bandsindicates successful sterilization, deimmunization, or disinfection;

FIG. 7 is a homology diagram comparing protein sequences of a researchClostridium species and two pathogenic Clostridium species;

FIG. 8 shows an example of a Western Blot for a defined Clostridiumsurrogate protein having a predetermined sequence that has beensubjected to sterilization, deimmunization, or disinfection;

FIG. 9 is a homology diagram comparing protein sequences of a threeresearch Bacillus species;

FIG. 10 shows an example of a Western Blot for a defined Bacillussurrogate protein having a predetermined sequence that has beensubjected to sterilization, deimmunization, or disinfection;

FIG. 11 is a homology diagram comparing protein sequences of threepathogenic Mycobacterium species;

FIG. 12 shows an example of a Western Blot for a defined Mycobacteriumsurrogate protein having a predetermined sequence that has beensubjected to sterilization, deimmunization, or disinfection;

FIG. 13 is a homology diagram comparing protein sequences of threepathogenic Staphylococcus species;

FIG. 14 shows an example of a Western Blot for a defined Staphylococcussurrogate protein having a predetermined sequence that has beensubjected to sterilization, deimmunization, or disinfection;

FIG. 15 is a homology diagram comparing protein sequences of a researchPseudomonas species and two pathogenic Pseudomonas species;

FIG. 16 shows an example of a Western Blot for a defined Pseudomonassurrogate protein having a predetermined sequence that has beensubjected to sterilization, deimmunization, or disinfection;

FIG. 17 is a homology comparing protein sequences of two pathogenicTrichophyton species;

FIG. 18 shows an example of a Western Blot for a defined Trichophytonsurrogate protein having a predetermined sequence that has beensubjected to sterilization, deimmunization, or disinfection;

FIG. 19 is a homology comparing protein sequences of four pathogenicCandida species;

FIG. 20 shows an example of a Western Blot for a defined Candidasurrogate protein having a predetermined sequence that has beensubjected to sterilization, deimmunization, or disinfection;

FIG. 21 is a homology comparing protein sequences of α-Gliadin from manyspecies of commonly consumed grains;

FIG. 22 shows an example of a Western Blot for a defined α-Gliadinsurrogate protein having a predetermined sequence that has beensubjected to sterilization, deimmunization, or disinfection.

DETAILED DESCRIPTION OF THE INVENTION

Aside from the preferred embodiment or embodiments disclosed below, thisinvention is capable of other embodiments and of being practiced orbeing carried out in various ways. Thus, it is to be understood that theinvention is not limited in its application to the details ofconstruction and the arrangements of components set forth m thefollowing description or illustrated in the drawings. If only oneembodiment is described herein, the claims hereof are not to be limitedto that embodiment. Moreover, the claims hereof are not to be readrestrictively unless there is clear and convincing evidence manifestinga certain exclusion, restriction, or disclaimer.

The method for rapidly determining effective sterilization,deimmunization, and/or disinfection of equipment or supplies of one ormore embodiments of this invention may be utilized to qualifysterilization, deimmunization, and/or disinfection by a device, e.g., asterilization device, a deimmunization device, or a disinfection deviceand provide improvements to the conventional methods discussed above. Inone example, the method for rapidly detecting effective sterilization,deimmunization, and/or disinfection of equipment or supplies of one ormore embodiments of this invention may be based on a specific measuringthe complete destruction of a specific protein critical for anorganism's growth and requires only a few hours to return absoluteresults. The method for rapidly detecting effective sterilization,deimmunization, and/or disinfection of equipment or supplies of one ormore embodiments of this invention also contains multiple layers ofinternal controls that enable a clear determination if either falsepositive and false negative results have occurred. This allows theavoidance of unnecessary repairs to sterilization, deimmunization, ordisinfection equipment and eliminates false negative tests and moredangerous exposure of patients to improperly sterilized, deimmunized, ordisinfected medical equipment or supplies.

The method for rapidly detecting effective sterilization,deimmunization, and/or disinfection of equipment or supplies of one ormore embodiments of this invention may be used to rapidly determine if asterilization, deimmunization, and/or disinfection device is effectivelydestroying specific infectious or immunogenic agents, defined herein asinfectious or pathogenic proteins, infectious spore forming bacteria,infectious vegetative bacteria, infectious fungus, infectious viruses,and immunogenic proteins.

In one embodiment, the method for rapidly determining effectivesterilization, deimmunization and/or disinfection of equipment and/orsupplies by a device, such as a sterilization device, e.g., by a devicethat applies cycles of a solvent and electromagnetic radiation, e.g.,microwaves, such as disclosed in U.S. application Ser. No. 15/330,469 bythe assignee hereof, hereinafter the '469 patent application, any of thesterilization devices discussed in the Background section above, thedevices for the methods of sterilization shown in Table 3 above, anautoclave, of similar type sterilization device known to those skilledin the art, a deimmunization device, e.g., a device that applies cyclesof a solvent aid electromagnetic radiation, e.g., microwaves, such asdisclosed in the '469 patent application and/or disinfection device,e.g., a device that applies cycles of a solvent and electromagneticradiation, e.g., microwaves, such as disclosed in the '469 patentapplication, or the devices for the methods of disinfection shown inTable 3 above, includes providing a defined surrogate protein having apredetermined sequence representative of an infectious agent potentiallycontaminating the equipment and/or supplies to be sterilized,deimmunized and/or disinfected by the device, step 100, FIG. 1. Thedefined surrogate protein having the predetermined sequence is thensubjected to sterilization, deimmunization, or disinfection, step 102.The effectiveness of the sterilization, deimmunization, and/ordisinfection by the device is then rapidly determined by determining ifthe defined surrogate protein having the predetermined sequence has beendestroyed, step 106, as discussed further below.

The method preferably directly measures the irreversible destruction ofthe defined surrogate proteins having the predetermined sequence thatare critical for survival and/or growth of such infections agents.

In one embodiment, the method utilizes one or more prion detectionindicator samples configured as a defined surrogate protein. In thisexample, the defined surrogate protein has the following predeterminedsequence:

(SEQ ID No: 9) KKRPKPGGWN TGGSRYPGQG SPGGNRYPPQ GGTWGQPHGG GWGQPHGGSW 50 GQPHGGSWGQ PHGGGWGQGG GTHNQWNKPS KPKTNLKHVA GAAAAGAVVG 100GLGGYMLGSA MSRPMIHFGN DWEDRYYREN MYRYPNQVYY RPVDQYSNQN 150NFVHDCVNIT IKQHTVTTTT KGENFTETDV KMMERVVEQM CVTQYQKESQ 200 AYYDGRRS 208

The defined prions surrogate protein having the predetermined sequenceabove for prion detection is then subjected to sterilization,deimmunization, or disinfection, by a device, e.g., a sterilizationdevice, a deimmunization device or a disinfection device, e.g., byconventional methods discussed in the Background section above, or byapplying cycles of a solvent and electromagnetic radiation, e.g.,microwaves, such as disclosed in the '469 patent application.

In this example, to rapidly determine the effectiveness ofsterilization, deimmunization, or disinfection of equipment or suppliesby the device, a determination is made if the defined surrogate proteinhas been destroyed using a sensitive protein analysis procedure, such asWestern Blot analysis, or similar protein analysis techniques, such asfluorescence analysis of proteins covalently crosslinked to solidsurfaces used in protein array analysis which are extremely sensitiveprocesses that measure both the amount of full length intact definedsurrogate protein having a predetermined sequence and the amount of thedestroyed or degraded defined surrogate protein, protein array analysis,magnetic separation analysis, peptide analysis, mass spectrometryanalysis, gas chromatography analysis, or similar type analysis. In oneexample, the defined surrogate protein includes a protein critical forstability, growth, and/or infectious capacity of an infectious agent.The defined surrogate protein may include a protein critical forstability, growth, and/or infectious capacity of a surrogate organism ofthe infectious agent.

One embodiment of the method for rapidly detecting effectivesterilization, deimmunization, and/or disinfection of this inventionincludes providing the defined surrogate protein having a predeterminedsequence that is based on the development of a synthetic recombinanttest protein sequence with high homology to a section of a proteinencoded by the suf I loci in the targeted pathogenic genus, e.g., one ormore or all of the genus shown in Table 2 above, and the development ofa monoclonal or polyclonal antibody that is able to detect the definedsurrogate recombinant protein using Western Blot or other similarprotein analysis techniques discussed above. In genera that form spores,such as Bacillus and Clostridium, the target protein encoded in the sufI loci or defined surrogate protein having the predetermined sequence ofthe tests forms the spore coat protein CotA. In non-spore forming genussuch as the bacteria Mycobacterium, Staphylococcus and Pseudomonas, orthe fungus Candida, the target protein encoded in the suf I loci ordefied surrogate protein of the tests forms the cell division protein(FtsP). In other genera of bacteria and fungus, the target protein orencoded defined surrogate protein having the predetermined sequence inthe suf I loci may have additional names but all carry the same proteinstructure of multicopper oxidase with 3 cupredoxin superfamily domains.

For each method of rapidly determining effective sterilization,deimmunization, or disinfection, specific for its unique genus, apredetermined quantity of the defined surrogate having the predeterminedprotein sequence is placed on a carrier, such as filter paper, or atube, or on the surface of an object, such as a glass or slide, amicrotiter plate, a flexible membrane, magnetic beads, e.g., magneticbeads used for magnetic bead separation or similar type object orsurface. For Western analysis, the defined surrogate protein having thepredetermined sequence is not covalently linked to the carrier orsurface. In other protein analysis processes such as protein array ormagnetic bead separation, the defined surrogate protein having thepredetermined sequence is covalently linked to the earner or solidsurface. After treatment with the sterilization, deimmunization ordisinfection equipment of process and Western analysis, this involvesrecovering the recombinant defined surrogate protein having thepredetermined sequence, both in its intact and fragmented forms, fromthe carrier, surface tube, or object, treating the sample withdenaturing loading buffer and running the sample on an acrylamide gel. Acontrol sample that was not subjected to sterilization, deimmunization,or disinfection is also included. In one example, the samples aretransferred nylon membrane and the intact and fragmented samples arevisualized using the specific antibody. If the sterilization,deimmunizing or disinfection equipment is operating correctly, thecontrol sample will have protein indicator bands that are easilyvisualized but the treated sample will be absent any type proteinindicator bands indicating the sterilization, deimmunizing ordisinfection equipment was able to irreversibly fragment the recombinantdefined surrogate protein. If other protein analysis processes such asprotein array or magnetic bead separation are used, the solid surface towhich defined surrogate protein having the predetermined sequence wascovalently linked will be tested such that intact defined surrogateprotein having the predetermined sequence can be visualized using thespecific antibody and fragment and/or destroyed defined surrogateprotein having the predetermined sequence is no longer detected by theprocess. As the amino acid sequence and structure of the definedsurrogate protein having a predetermined sequence is highly homologousto the target protein in the pathogenic members of the genus,irreversible destruction of the defined surrogate protein indicates thatthe sterilization, deimmunizing or disinfection equipment will have alsodestroyed the target proteins, resulting in destruction of all membersof the genus that may be on the sterilization, deimmunizing ordisinfection equipment or supplies.

In accordance with one or more embodiments of the method for rapidlydetermining effective sterilization, deimmunization, and/ordisinfection, the predetermined sequence of the defined surrogateprotein and the corresponding peptide used for development of apolyclonal antibody for Western Blot analysis include one or more of thefollowing predetermined sequences:

For Clostridium:

(SEQ ID NO: 1)         10         20         30         40MYNYTSAKYE VPIAIQDRSF NEDGSLNFPS EGDNPTIHPY         50WQPEFFGDTI MVNGRVWPNM NVDMTRYRFR LLNGSNARFYNLKFSNSMQF WQIGTDGGYL NKPVPLTSLL ISPGERADILVDFTEIPAGT RIILNNDANA PYPTGDAPDK DTTGQIMQFT VQHNDHHHHH H

The peptides used for the development of polyclonal and monoclonalantibodies for use by Western Blot analysis for the above sequence are:

KYEVPIAIQDRSFNEDGSLNFPSE (SEQ ID NO: 10) and YLNKPVPLTSLLISPGERADILVD(SEQ ID NO: 11)For Bacillus:

(SEQ ID NO: 2)         10         20         30         40MTLEKTYYEV TMEECTHQLH RDLPPTRLWG YNGLFPGPTI         50EVKRNENVYV KWMNNLPSTH FLPIDHTIHH SDSQHEEPEVKTVVHLHGGV TPDDSEGYPE AWFSKDFEQT GPYFKREVYHYPNQQRGAIL WYHDHAMALT RLNVYAGLVG AYIIHDPKEK RLKHHHHHH

The peptides used for the development of polyclonal and monoclonalantibodies for use by Western Blot analysis for the above sequence are:

QRGAILWYHDHAMALTRLNVYAGL (SEQ ID NO: 12) and QLHRDLPPTRLWGYNGLFPGPTIE(SEQ ID NO: 13)For Mycobacterium:

(SEQ ID NO: 3)         10         20         30         40MTGMPEGEGV DSNLLGGDGG DIAYPYYLIN GRIPVAATSF         50KAKPGQRIRI RIINSAADTA FRIALAGHSM TVTHTDGYPVIPTEVDALLI GMAERYDVMV TAAGGVFPLV ALAEGKNALA RALLSTGAGS PPDHHHHHH

The peptides used for the development of polyclonal and monoclonalantibodies for use by Western Blot analysis for the above sequence are:

DTAFRIALAGHSMTVTHTDGYPVIPTEVD (SEQ ID NO: 14) and     VFPLVALAESKNALARALISTGAGS (SEQ ID NO: 15)For Staphylococcus:

(SEQ ID NO: 4)         10         20         30         40MTGYKNYTLK AQKGKTEFYK NNFSNTLGYN GHLIGPTLKL         50KKGDKVKIKL INNLDENTTF HWHGLEVNGK VDGGPSQVIKPGKEKTIKFE VNQDSATLWY HPHPSPNTAK QVYNGLSGLL YIEDSKKNHH HHHH

The peptides used for the development of polyclonal and monoclonalantibodies for use by Western Blot analysis for the above sequence are:

NFSNTLGYNGNLLGPTLKLKKGDKVKIKL (SEQ ID NO: 16) andKFEVNQDSATLWYNPHPSPNTAK (SEQ ID NO: 17)For Pseudomonas:

(SEQ ID NO: 5)         10         20         30         40MTGFRHEKVL CLKTWHVDEQ GAFTPFSVPR QAAREGTRGR         50YSTINGKHVP TIDLPAGQIV RVRLLNVSNT VTYRLNPNGEARIYAVDGHP VEPRGFEGQY WIGPGMRLEL ALKVPEAGTE LSLRDGPVRL ATIRSVAHHH HHH

The peptides used for the development of polyclonal and monoclonalantibodies for use by Western Blot analysis for fee above sequence are:

DLPAGQIVRVRLLNVDNTVTYRLN (SEQ ID NO: 18) and QYWIGPGMRLELALKVPEAG(SEQ ID NO: 19)For Trichophyton:

(SEQ ID NO: 6)         10         20         30         40MTITLEWSVT TGYRRLDGVK KRVYLINGLF PGPTIEARSG         50DSLQVQVTNN IQDEGLVIHW HGLHMRGANH MDGVTGVTQCPIVPGDSMLY NFTISQSQSG TFWYHAMSAL QRAEGLYGGF VVHKPSTHHH HHH

The peptides used for the development of polyclonal and monoclonalantibodies for use by Western Blot analysis for the above sequence are:

YRRLDGVKKRVYLINGLFPGPTIE (SEQ ID NO: 20) and TQCPIVPGDSMLYNFTISQSQSG(SEQ ID NO: 21)For Candida:

(SEQ ID NO: 7)         10         20         30         40MTAETHTWYF KTSWVDANPD GVFPRKMIGF NDSWPLPTLR         50VKKGDTVNLY LINGFDDRNT SLHFHGLFQH GTNQMDGPEMVTQCPIPPGE TFLYNFTVDD QVGSYWYHSH TSGQYGDGMR GVFIIEDHHH HHH

The peptides used for the development of polyclonal and monoclonalantibodies for use by Western Blot analysis for the above sequence are:

GFNDSWPLPTLRVKKGDTVNLYL (SEQ ID NO: 22) and WYFKTSWVDANPDGVFPRKMIG(SEQ ID NO: 23)For α-Gliadin:

(SEQ ID NO: 8)         10         20         30         40MKTVRVPVPQ PQPQNPSQPQ PQRQVPLVQQ QQFPGQQQQF         50PPQQPYPQPQ PFPSQQPYLQ LQPFPQPQPF PPQLPYHHHH HH

The peptide used for the development of monoclonal or polyclonalantibody used by Western Blot analysis for the above sequence is:

FPPQQPYPQPQPFPSQQPYLQLQPFPQPQ (SEQ ID NO: 24)

Western Blot analysis typically utilizes equipment, e.g., acrylamidegel, a power supply to create an electric field to trigger proteinmigration where smaller fragments move faster than larger fragments toseparate intact proteins from fragmented or degraded proteins, amembrane, transfer equipment, and visualization equipment. Western Blotanalysis also preferably utilizes specific reagents, e.g., a positivecontrol protein to show the location of a full length defined surrogateprotein and specific antibody for the defined surrogate protein, e.g.,any of the defined surrogate proteins having the associatedpredetermined sequences and the corresponding antibody above. Thespecific antibody binds to the associated defined surrogate protein,both full length, and fragments, to provide a visualization if thedefined surrogate protein was destroyed by sterilization,deimmunization, or disinfection.

Western Blot analysis for infectious organisms may also typicallyinclude a defined number of colony forming units (CFU) or spores of thetest pathogen which are added to a stable substrate such as filter paper(dried) or other sample holder. The number of CFU or spores will containa defined quantity of each indication protein to be followed.

Western Blot analysis may also be conducted on defined surrogateproteins having the associated predetermined sequences samples placed onindicator strips or other small sample holders that maybe subjected tosterilization, deimmunization or disinfection. After sterilization,deimmunization or disinfection, the defined surrogate proteins from theindicator strips or other small sample holders are extracted from thefilter paper or other sample holders into a loading dye and denatured(eliminating any tertiary protein structure). The samples are then runon the gel, including control wells with (1) size marker, (2) positivecontrol protein, (3) other controls if needed. After transfer tomembrane, defined surrogate proteins and protein fragments arevisualized with unique antibodies preferably having a high affinity andspecific binding to an indicator region of the protein being tested.Successful sterilization, deimmunization or disinfection will result inthe loss of all indicator proteins of defined length. The developmentprocess may require side by side Western Blots and standard growthstudies to demonstrate sufficient equivalence.

Western Blot analysis may also require defining the defined surrogateproteins having the associated predetermined sequence, positive controlproteins, and/or a negative control proteins and may require thedevelopment of a polyclonal or monoclonal antibodies that specificallybind to specific defined surrogate proteins discussed above in WesternBlot analysis or other protein analysts format, both full-length andfragments, and the positive control protein. The antibody will not bindto the negative control protein. It is also possible that a secondaryantibody that is labeled with an enzyme or other visualization markerwill be needed to visualize the detection antibody.

Protein array analysis can be used as a substitution for Western blotanalysis in circumstances in which less sensitivity can be tolerated,but faster results are needed. In a Western Blot analysis, a substratesuch as a piece of filter paper or tube is used to temporarily hold therecombinant protein sample during testing of sterilization equipment.After sterilization, deimmunization and/or disinfections the recombinantprotein sample is removed from the substrate, denatured, separated on anacrylamide gel and transferred to nylon membrane before visualizing withantibodies. In protein array analysis, the recombinant protein sample,e.g., one or more of the defined surrogate proteins having theassociated predetermined sequence discussed above, is covalentlycrosslinked to solid surfaces such as glass, plastics or metal beads.After treatment by sterilization, deimmunization or disinfection, thesolid surfaces, with the covalently crosslinked recombinant proteins aredirectly visualized with antibodies. Unlike Western blot analysis thatcan visualize the amount of protein fragmentation has occurred, proteinarray analysis can indicate how much of the protein sample has lostregions that are recognized by visualizing antibodies protein assayanalysts but is unable to determine what other areas of the recombinantproteins were not destroyed. Thus, protein array analysis is highlysuitable for testing the presence or absence of intact test proteins,such as the defined surrogate proteins having the associatedpredetermined sequence homologous to proteins critical for spore coatsor bacteria or fungus survival or growth, e.g., one or more of thedefined surrogate proteins having the predetermined sequences forClostridium, Bacillus, Mycobacterium, Staphylococcus, Pseudomonas,Trichophyton, Candida, and α-Gliadin show above. Because protein assayanalysis cannot determine the absolute level of protein fragmentationthat has occurred, protein array analysis may not be sensitive enoughfor quantifying the complete destruction of infectious proteins likeprions although it is not excluded as a test method if less sensitivityis tolerated.

To establish a protein array analysis, defined quantities of the definedsurrogate proteins for Clostridium, Bacillus, Mycobacterium,Staphylococcus, Pseudomonas, Trichophyton, Candida, and α-Gliadin aboveare covalently linked to a solid surface. The solid surface may includea wide support surface, such as a microscope slide made of glass orsilicon, a flexible membrane, a magnetizable bead, a microtiter plate,or other similar solid surface to which a selection of the definedsurrogate proteins having the associated predetermined sequence can bepermanently attached. The surface used is determined by the typedetector that will be used to determine the quantity of protein left onthe support after sterilization, deimmunization or disinfection. Aftersterilization, deimmunization or disinfection, the solid surface isvisualized with antibodies or other specific ligands that willspecifically bind to the intact defined surrogate protein but not todestroyed defined surrogate proteins. The antibodies or other ligandscan contain fluorescent dyes or other detection enabling attachments.Control slides that were not subjected to sterilization, deimmunizationand/or disinfection are included as a positive control to enableestimations for the relative amount of test protein destroyed bysterilization, deimmunization and/or disinfection. FIG. 2 shows anexample of multi-well glass slide 130 which has the control definedsurrogate protein having the associated predetermined sequence in thewells exemplary, indicated at 132 and multi-well glass slide 134 whichhas one of the defined surrogate proteins having the predeterminedsequence for Clostridium, Bacillus, Mycobacterium, Staphylococcus,Pseudomonas, Trichophyton, Candida, and α-Gliadin in the wells,exemplarily indicated at 136. In this example, control multi-well glassslide 130 was not subject to sterilization, deimmunization, ordisinfection and visualization of the antibodies is indicated by theshading as shown which indicates the defined surrogate protein havingthe associated predetermined sequence was not sterilized, deimmunized,or disinfected. In this example, multi-well glass slide 134 was subjectto sterilization, deimmunization and/or disinfection and visualizationshows no presence of antibodies and, therefore, the defined surrogateproteins having the predetermined sequence were effectively sterilized.Thus, in this embodiment, the method for rapidly determining effectivesterilization was able to quickly determine effective sterilization,deimmunization and/or disinfection in as little as two hours dependingon the type of detection equipment used to rapidly determine effectivesterilization, deimmunization or disinfection discussed above.

One key benefit of using protein array analysis in accordance with oneembodiment of the method for rapidly determining effectivesterilization, deimmunization, and/or disinfection of this invention isthe covalently linked surrogate proteins on the surface of an object canbe used for testing sterilization, deimmunization or disinfection by asterilization, deimmunization or disinfection device that may useradiation or heat, as well as devices that use flowing disinfectants orgases to sterilize, deimmunize or disinfect medical equipment orsupplies. In one example, holder 150, FIG. 3, may be used to holdmultiwall glass slide 152 having one or more wells, exemplarilyindicated at 154, which each holds one or more samples of the definedsurrogate protein having the predetermined sequence discussed above,e.g., one of Clostridium, Bacillus, Mycobacterium, Staphylococcus,Pseudomonas, Trichophyton, Candida or α-Gliadin. Holder 150 withmulti-well glass slide 152 including one or more samples of the definedsurrogate protein having the predetermined sequence discussed above maythen be placed below flow 156 of sterilization, deimmunization, ordisinfection agent at the end of a medical device undergoingsterilization, deimmunization, or disinfection, indicated at 158. Afterthe test is complete, holder 150 is then removed, indicated at 160, andmulti-well glass slide 152 can be easily transferred for completeprotein analysis and for rapid detection of sterilization,deimmunization, or disinfection as discussed above.

If magnetizable beads are used as the solid surface, one or more of thedefined surrogate proteins having the associated predetermined sequencecould also be mixed into liquid sterilization, deimmunization and/ordisinfection steam and later harvested with a strong magnet. Flowingdisinfectants or gases would result in the complete loss of the definedsurrogate protein having the associated predetermined sequence samplesthat are not covalently linked to a support surface and the proteinarray analysis may be used to rapidly determine effective sterilization,deimmunization or disinfection.

Both Western Blot Analysis and protein array analysis are an indirectmeasurement of sterilization, deimmunization and/or disinfection.Sterilization is the complete destruction of spore forming infectiousorganisms so the standard direct measurements are long growth studies todetermine the number of organisms that survived the sterilizationprocess. Deimmunization is the complete destruction of immunogenicproteins such that no protein fragments capable of triggering an immunereaction in human or animal remain. Disinfection is the completedestruction of vegetative bacteria, fungus and/or viruses. As disclosedherein, die defined surrogate proteins are considered to be in the formof isolated fragments and destroyed if proteins critical for thesurvival of the infectious agents are used in quantities 10,000 to1,000,000 times as much as would occur in a standard sample of intactinfectious agents. To correctly establish the scale to determine successratio between the direct and indirect tests, multiple conditions arepreferably utilized to determine the Western Blot analysis and proteinarray analysis conditions that perfectly align with standardsterilization, deimmunization and/or disinfection studies using intactinfectious organisms and post sterilization growth conditions.

The samples prepared for Western Blot analysis and protein arrayanalysis may use a wide range of circumstances. If the sterilization,deimmunization and/or disinfection equipment uses flowing gases orliquid to wash over the surfaces to he sterilized holder 150, FIG. 3,and multi-well glass slide 152 or similar type device, may be used toensure the defined surrogate protein samples remain In the flowingstream of disinfecting gas or liquid. For example, if the sterilization,deimmunization or disinfecting gas or liquid is washed through medicalequipment to sterilize, deimmunize, or disinfect the inside of a lumen,holder 150 with the multi-well glass slide 150 having one or moredefined surrogate proteins having the associated predetermined sequencetherein, e.g., Clostridium, Bacillus, Mycobacterium, Staphylococcus,Pseudomonas, Trichophyton, Candida, and α-Gliadin analysis tests couldbe connected to the end of the stream to better measure the quality ofsterilization, deimmunization, or disinfection that occurred through theentire length of the medical equipment lumen.

The following examples are exemplary and explanatory only and do notlimit or restrict this invention.

EXAMPLES Example 1: Comparing Amino Acid Sequence of Human PrP Proteinto Other Species

It is important to qualify the ability of a sterilization device todestroy prions that may be contaminating medical equipment. For thespecific test described herein in accordance with one or moreembodiments of the method for rapidly determining effectivesterilization, deimmunization, or disinfection of this invention, adefined quantity of the defined surrogate prion protein discussed aboveis provided and then evaluated using Western Blot analysis and anantibody specific for the defined prion surrogate protein.

To protect the human operators of the test, infectious human prionscannot be used. Instead, the defined surrogate protein is used thatincorporates all the characteristics of human prion (PrP) proteins, withthe critical exception that it cannot infect humans. In this example, toselect the defined surrogate PrP protein to be used, a protein analysiswas conducted comparing the amino acid protein sequence of human PrPprotein against a selection of the protein sequence databases of otherspecies including a primate, two companion animals, two food animals andtwo research animals. From this analysis, the mouse PrP protein wasdetermined to be the best candidate surrogate protein. Structurally,mouse PrP protein is as robust as the human PrP protein and thus will beequivalently resistant to a wide range of destructive methods but isalso sufficiently different as to be unable to infect humans. Arrow 168,FIG. 4, indicates the initiation location for Pr27-30, the smallest PrPfragment that retains infectivity. If a PrP protein is cleaved anywherefrom this initiation point to the end of the protein sequence, theresulting fragments can no longer cause disease.

To evaluate additional surrogate PrP proteins that could be used, themouse PrP protein sequence was compared against the protein sequencedatabase. Table 5 below shows the wide diversity of a predetermined listof species PrP proteins that could be used as well as each protein'ssequence ID and its homology to Mouse PrP. The human sequence is lessthan 90% homologous to mouse PrP. The search also demonstrates howconserved the PrP protein is across a wide range of mammalian speciescontained within the sequence database and any one of these could beused as the surrogate protein in the Protein Indicator Test. Thedatabase also contains the sequence for chicken PrP but as it is lessthan 50% homologous to other mammalian PrP proteins, it is possible thatit or other related proteins could be used in the Protein Indicator Testbut its divergence could impact its resistance to destruction. Thiswould make it a less suitable surrogate for the test than otherproteins, especially any mammalian PrP protein.

TABLE 5 Potential PrP Protein that could be used as Surrogate Protein inSterilization Indication Tests. Information about each PrP proteinincludes species, Sequence ID number and homology to Mouse recombinantPrP protein. Homology to Species Sequence ID Number Mouse (%) Mouse (Musmusculus) sp|P04925.2|PRIO_MOUSE 100 Rat (Rattus norvegicus)sp|P13852.2|PRIO_RAT 98 Cotton Rat (Sigmodon hispidus)sp|Q9Z0T3.1|PRIO_SIGHI 97 Chinese Hamster (Cricetulus griseus)sp|Q60506.1|PRIO_CRIGR 97 Grey Dwarf Hamster (Cricetulus migratoriussp|Q60468.1|PRIO_CRIMI 95 Golden Hamster (Mesocricetus auratus)sp|P04273.1|PRIO_MESAU 95 Greater Kudu (Tragelaphus strepsiceros)sp|P40243.1|PRIO2_TRAST 90 Red-bellied Titi (Callicebus moloch)sp|P40248.1|PRIO_CALMO 96 Three-striped Night Monkey (Aotus trivirgatus)sp|P40245.1|PRIO_AOTTR 94 Black-capped capuchin (Sapajus apella)sp|P40249.1|PRIO_CEBAP 95 Common Marmoset (Callithrix jacchus)sp|P40247.1|PRIO_CALJA 95 Red-faced Spider Monkey (Ateles paniscus)sp|P51446.1|PRIO_ATEPA 94 Geoffrey's Spider Monkey (Ateles geoffroyi)sp|P40246.1|PRIO_ATEGE 91 Nilgai (Boselaphus tragocamelus)sp|Q5UJG7.1|PRIO_BOSTR 86 Alpine Musk Deer (Moschus chrysogaster)sp|Q68G95.1|PRIO_MOSCH 88 Common Squirrel Monkey (Saimiri sciureus)sp|P40258.1|PRIO_SAISC 91 Gelada Baboon (Theropithecus gelada)sp|Q95270.1|PRIO_THEGE 90 Black Crested Mangabey(Lophocebus aterrimus)sp|P67990.1|PRIO_LOPAT 90 Mona Monkey(Cercopithecus mona)sp|P61761.1|PRIO_CERMO 91 Patas Monkey(Erythrocebus patas)sp|Q95174.1|PRIO_ERYPA 91 Grivet(Chlorocebus aethiops)sp|P67988.1|PRIO_CHLAE 90 Mantled Guereza(Colobus guereza)sp|P40251.1|PRIO_COLGU 91 Bornean orangutan(Pongo pygmaeus)sp|P40256.1|PRIO_PONPY 91 François' Langur (Trachypithecus francoisi)sp|P40257.2|PRIO_TRAFR 91 Sooty Mangabey (Cercocebus atys)sp|Q95176.1|PRIO_CERAT 91 Crab-eating Macaque (Macaca fascicularis)sp|P67992.1|PRIO_MACFA 91 Mandrill(Mandrillus sphinx)sp|P40255.1|PRIO_MANSP 91 Gorilla (Gorilla gorilla gorilla)sp|P40252.1|PRIO_GORGO 90 Cat (Felis catus) sp|O18754.3|PRIO_FELCA 87Human (Homo sapiens) sp|P04156.1|PRIO_HUMAN 89 Lar Gibbon(Hylobates lar)sp|P61766.1|PRIO_HYLLA 89 Bighorn Sheep (Ovis canadensis)sp|Q7JIH3.1|PRIO_OVICA 88 Goat(Capra hircus) sp|P52113.1|PRIO_CAPHI 88Blackbuck(Antilope cervicapra) sp|Q5UJG1.1|PRIO_ANTCE 85 Takin(Budorcastaxicolor) sp|Q95M08.1|PRIO_BUDTA 87 Sheep (Ovis aries)sp|P23907.1|PRIO_SHEEP 87 Rabbit (Oryctolagus cuniculus)sp|Q95211.1|PRIO_RABIT 90 Water Buffalo (Bubalus bubalis)sp|Q5UJH8.1|PRIO_BUBBU 84 Rocky Mountain Elk sp|P67986.1|PRIO_CEREN 87(Cervus canadensis nelsoni) Mule Deer (Odocoileus hemionus)sp|P47852.1|PRIO_ODOHE 87 Lesser Kudu (Tragelaphus imberbis)sp|Q5UJG3.1|PRIO_TRAIM 85 Greater Kudu(Tragelaphus strepsiceros)sp|P40242.1|PPIO1_TRAST 84 Cow (Bos taurus) sp|P10279.2|PRIO_BOVIN 84Common Brushtail Possum sp|P51780.1|PRIO_TRIVU 77 (TrichosurusVulpecula) Giant Panda(Ailuropoda melanoleuca) sp|Q6EH52.1|PRIO_AILME 84American Mink (Neovison vison) sp|P40244.1|PRIO_NEOVI 79 Dromedary Camel(Camelus dromedaries) sp|P79141.1|PRIO_CAMDR 88 Pig (Sus scrofa)sp|P49927.1|PRIO_PIG 79 Ferret(Mustela putorius furo)sp|P52114.1|PRIO_MUSPF 78 Artic Fox(Vulpes lagopus)sp|B0FYL5.1|PRIO_VULLA 78 Dog (Canis lupus familiaris)sp|O46501.1|PRIO_CANFA 85 Chicken (Gallus gallus) sp|P27177.2|PRIO_CHICK43

Example 2: Developing Method for Testing the Ability to Destroy PrPProteins

One purpose of developing the defined prion surrogate protein test is toprovide a rapid method for determining effective sterilizationequipment. The method preferably has multiple steps including atleast: 1) preparing defined prion surrogate proteins having apredetermined sequence, 2) subjecting the prion surrogate proteinsamples to sterilization, and 3) using Western Blot analysis tovisualize the effects of sterilization of defined prion surrogateprotein samples. Successful sterilization has occurred when all of thedefined prion surrogate protein sample was fragmented and, as a resultof the protein fragmentation, none remains to bind to the visualizationantibodies. If sterilization was not successful, protein bands will besee on the Western Blot analysis.

A defined surrogate protein having a sequence above for the prions,recombinant mouse PrP full-length protein (208 amino acids long) wasobtained from Abcam, Cambridge Mass. On a Western gel, the recombinantprotein runs at about approximated 28 kDa in size, indicated at 290,FIG. 5.

The Western Blot analysis discussed above is well-established and is anextremely sensitive method to determine the presence and/or absence of aspecific protein. The first step in Western Blot analysis involvespreparing the defined prion surrogate protein samples for separation bysize. In accordance with one embodiment of the method for rapidlydetermining effective sterilization, deimmunization, or disinfection ofthe defined prion surrogate proteins will be run as single denaturedproteins and thus must be denatured through the use of a denaturingloading buffer, boiled for 10 minutes and then run on a denaturing gel.Once loaded on to an appropriate acrylamide gel, the samples aresubjected to electric current that results in the protein fragmentstraveling at different rates depending on size with smaller fragmentsmoving faster than larger ones. In one example, the gel contained 8%acrylamide. To be able to monitor the separation of the defined prionsurrogate protein samples, a standard sample with a mix of proteins ofdefined sizes was also included. For some experiments discussed herein,additional protein samples may be included to provide for controls ofthe sample handling conditions.

After separation, the defined prion surrogate protein samples aretransferred to a special nylon membrane before being permanentlycross-linked to the membrane. The final steps include incubating thenylon membrane with a primary antibody available from Abcam, CambridgeMass. that specifically binds to the defined surrogate protein ofinterest. In the experiments discussed herein, the primary anti-PrPantibody was a rabbit monoclonal antibody that was raised against asynthetic peptide corresponding to residues near the C-terminus of humanPrion protein PrP, as indicated by underlined region indicated at 170,FIG. 4. In addition to binding to both PrP_(c) and PrP_(sc), theantibody will bind to the infectious protein form PrP 27-30. The primaryantibody binds to Human, Rat and Mouse PrP proteins. The secondaryantibody is a HRP-labelled goat anti-rabbit available from Abcam,Cambridge Mass. to enable visualization of the infectious PrP fragments,both intact and fragmented. If the defined prion surrogate proteins havebeen fragmented into non-infectious fragments that eliminate theC-terminus region of the PrP, the antibody will not bind them. Withoutthis C-terminus region, the degraded PrP protein is no longerinfectious. Very small fragments and amino acids which are no longerinfectious will be too small to be retained on the gel.

The first tests involved carefully drying defined quantities ofrecombinant mouse PrP protein onto filter papers to assist in storage,transport to and from location of sterilization equipment and handlingduring sterilization tests. To create, test strips of filter paper werecut 4 mm wide by 20 mm long and the bottoms were squared. Serialdilutions (1:3) of mouse PrP protein were created to contain proteinsolutions 0.1 μg/μl to 0.0037 μg/μl. The protein preps were addeddropwise to the filler paper strips until 10 ul were added. The filterpaper strips were then air dried. Filter paper strips were prepared induplicate with 1 μg, 0.333 μg, 0.111 μg, or 0.033 μg appliedrespectively to a pair of strips. When dry, the strips were placed inEppendorf tubes and 50 ul 1× Loading Buffer where BME was added. Thestrips and loading buffer were incubated at 95° C for 5 minutes thengiven a quick spin in a micro centrifuge. For one of each paired sample,the filter paper strip was taken out of the Eppendorf tube and insertedinto the well of the SDS-PAGE gel. For these samples, electroporationwas used to extract any residual defined prion surrogate protein thatwas retained on the filter paper after extraction by boiling andcentrifugation. Half of the loading buffer from these tubes was alsoloaded into the same well. For the other samples of the pair onlyboiling and centrifugation was used to extract the defined prionsurrogate protein front the filter strips. From these, half of theloading buffer was added to the wells without also adding the filterpaper. A control sample of 0.5 μg protein (equivalent to half of the 1μg concentration samples) was also loaded on the gel for comparison. Thegel was run and the Western Blot Analysis performed using the proceduredescribed above.

The results demonstrated that there was no significant differencebetween the protein concentrations that were dried onto the filter paperand the control sample. There was also no significant difference betweenthe extraction process using only boiling and centrifugation and themore difficult process of using boiling, centrifugation andelectroporation for extraction. In addition, the Western Blot analysisdemonstrated that the Western Blot analysis was sensitive to below 0.05μg. Hence, a 1 μg of recombinant mouse PrP protein should be used forall sterilization tests. The samples should be extracted from filterpaper using boiling in loading the buffer with BME and centrifugationand half of the sterilized samples used for Western Bolt analysis.

Example 3: Evaluation of Destruction of Prion Proteins UsingSterilization Equipment

Experiments were conducted to demonstrate a combination of one or moreof heat, a vaporizing solvent, and electromagnetic radiation, e.g.,microwaves, e.g., as disclosed in the '469 patent application, couldirreversibly destroy the defined prion surrogate protein shown with thesequence shown above. The stable PrP protein was selected for theexperiments as it cannot be irreversibly destroyed using a standardsterilization autoclave device. For the experiments, samples werecreated that each contained 1 μg of a structurally robust mouse PrPprotein and wrapped in 100% cotton paper to avoid extraneouscontamination. This containment was placed in a second layer of 100%cotton paper to increase stability during treatment. The samples weretreated with different temperatures and for differing numbers ofmoisture saturation and microwave cycles, e.g., as disclosed in the '469patent application. After treatment, the samples were prepared inloading buffer and boiled. Half of each sample was then run on adenaturing protein gel. After Western Blot analysis, it was possible tosee that certain combinations of temperature and treatment cyclescompletely destroyed the stable protein samples, shown by the absence ofprotein bands of gel indicated at 294, 296, FIG. 6. Other treatmentconditions did not destroy the stable proteins.

Example 4: Evaluation of Destruction of Prion Proteins Contained withinPolypropylene Tubes

Additional experiments were conducted to demonstrate that with thecombination of one or more of heat, saturating moisture and microwavesas disclosed in the '469 patent application could destroy the definedsurrogate stable PrP proteins if the samples were contained within avessel such as a polypropylene tube, in this example, samples of thedefined prion surrogate proteins were created that each contained about1 μg of a structurally robust mouse protein. However, instead of dryingthe protein samples onto filter paper, the 1 μg samples were driedwithin polypropylene tubes of different lengths including 0.75 cm and3.5 cm in length. The tubes were closed at one end (on to which thesamples were dried) and open at the other. The tubes were containedwithin 2 layers of 100% cotton paper to prevent cross contaminationduring treatment. The samples were subjected to sterilization by acombination of one or more of heat, a solvent, e.g., reverse osmosis(RO) filtered water, and electromagnetic radiation with differingnumbers of moisture saturation and radiation cycles, e.g., as disclosedin the '469 Patent Application. After treatment, the samples wereprepared in loading buffer and boiled. Half of each sample was then runon a denaturing protein gel. After Western blot analysis, it waspossible to see that certain combinations of temperature and treatmentcycles completely destroyed the stable protein samples (as demonstratedby the absence of protein bands of gel). Other treatment conditions didnot destroy the stable proteins. The results of the sterilizationtreatment of PrP samples dried onto filter paper and dried within apolypropylene tube were identical indicating that the method ofcontaining the PrP sample did not alter the results.

Example 5: Comparison of Prion Protein Indicator Test Results and MouseModel Prion Tests

All mice were kept in an AAALAC-accredited facility and handled incompliance with guidelines provided by the US Guide for the Care and Useof Laboratory Animals.

Creating Prion Infected Brain Homogenate: Brains from terminally illC57BL/6 mice infected with 22L prions were prepared as follows: eachbrain was homogenized (10%, w/v) in phosphate-buffered saline (PBS) byrepeatedly passing the material first through an 18-gauge needle andthen repeatedly through a 26 gauge needle. The brain homogenates werecombined to make a stock preparation, diluted with PBS (1/10), aliquotedinto 100 ul preps in 2 ml polypropylene freezing vials and frozen at−80° C. until use.

Division into treated and non-treated preps: frozen prion preps havingthe defined prion surrogate protein were allowed to thaw and the capsremoved. In duplicate, samples were sterilized by a combination of oneor more of heat, microwaves and saturating moisture, e.g., as disclosedin the '469 patent application. The used conditions were similar to theprocess disclosed in Example 4 above. In one example, the sterilizationpreferably includes: (A) 140° C., 100 cycles of microwave; (B) 100° C.,100cycles of microwave; and (C) room temperature, 100 cycles with nomicrowaves. The Prion Protein Indicator Test having the definedsurrogate protein samples were analyzed by Western Blot analysis asdescribed in Example 4 above. Depending on treatment conditions, thetest sample was completely destroyed (A), partially destroyed (B), orcompletely intact (C).

Testing infectiveness of Prion Prep: C57BL/6 mice aged 4-5 weeks weredivided into 4 cohorts, 10 mice per cohort, to receive the samples thatcorrelated with the following Prion Protein Indicator Test sampleshaving the defined prion surrogate protein; (A) complete destruction;(B) partial destruction; (C) no treatment and (D) PBS control. Afteranesthesia each mouse was intracerebrally inoculated with a 20ul-aliquot of the designated inoculum. The mice were observed up to oneyear after inoculation, unless they displayed terminal symptoms of PrPinfection including persistent signs of ataxia, kyphosis, somnolence,and hind leg weakness. Terminally-ill mice were euthanized and theirbrains divided sagittally along the midline and place formalin fixationfor histological analysis or flash-frozen in liquid nitrogen for proteinanalysis. At one year, all remaining mice (showed no obvious signs ofneurologic disease) were euthanized and their brains also divided forhistological analysis or protein analysis.

Results: Over the observation period, none of the mice that receivedeither the PBS control (D) or the brain homogenate treated with theconditions that demonstrated complete destruction on the Prion ProteinIndicator Test having the defined prion surrogate protein (A)demonstrated any symptoms of disease. After euthanization, none of thebrains demonstrated any signs of prion disease. Western Blot analysis ofthe brains showed no increase in concentrations of PrP proteins overnormal levels. The mice that received the brain homogenate and alsoreceived no sterilization treatment (C) or partial destruction (B) asindicated by the Prion Protein Indicator Test all demonstrated terminalsymptoms of PrP infection before the completion of the 1 yearobservation period. Their brains demonstrated obvious signs of priondisease and by Western analysis, the concentration of PrP proteins weregreatly increased over normal levels. Together the results of the mousestudy indicated a clear correlation between the results from the PrionProtein Indicator Test results and the mouse model results.

Example 6: Comparing Amino Acid Sequence of Multiple Members of theClostridium Genus

It is important to qualify the ability of a sterilization device, adeimmunization device, or a disinfection device to destroy bacteria ofany Clostridium species that may be contaminating medical equipment orsupplies. In this example, a defined quantity of the defined surrogateprotein having the predetermined SEQ ID NO: 1 discussed above wassubjected to sterilization, deimmunization, or disinfection to rapidlydetermine the effectiveness of the sterilization, deimmunization, ordisinfection using Western Blot analysis, protein array analysis, orsimilar type analysis, and the antibody for the defined Clostridiumsurrogate protein shown above. In this example, to protect the humanoperators of the test, the defined surrogate protein needs toincorporate critical characteristics of Clostridium proteins that arecritical for the survival and growth of members of the Clostridium genuswhile avoiding organisms that can infect humans.

To design the synthetic defined surrogate, Clostridium protein havingthe predetermined sequence, SEQ ID NO: 1, a protein analysis wasconducted comparing the amino acid sequences of the suf I loci gene frommultiple species of the Clostridium genus shown in Table 2 above. Inthis example, the Sequence IDs (found in Pubmed,www.ncbi.nlm.nih.gov/Pubmed/) for the suf I loci proteins, multipleClostridium species, used for the comparative are shown in Table 5below:

TABLE 5 (Clostridium). Sequences used to determine regions of highhomology in suf I locus of multiple Clostridium Species. SpeciesSequence ID Clostridium sporogenes WP_061905762.1 Clostridium botulinumWP_011948579.1 Clostridium novyi WP_039217212.1

The protein produced by the suf I loci is fundamental to survival andgrowth of a wide range of spores, bacteria and fungus. In species of theClostridium genus, the protein product of the suf I loci is called cotAand is critical for many live stages including strongly contributing tothe stability of the spore coat.

To design the defined Clostridium surrogate protein having SEQ ID NO: 1,the specific amino acids from the proteins listed in Table 5 werealigned to determine amino acid sequence regions that are highlyhomologous in all evaluated Clostridium species as shown in FIG. 7. Theprotein encoded by the suf I loci includes three cupredoxin domains thatare indicated at 200 for domain 1, 202 for domain 2, and 204 for domain3. In the Clostridium genus, domain 2, indicated at 202, shows highhomology between Clostridium species so was used to for the design ofthe synthetic surrogate protein SEQ ID NO: 1 above to be created for theClostridium test and the corresponding peptides discussed above wereused to develop the polyclonal and monoclonal antibodies for use byWestern Blot analysis.

Example 7: Developing Western Test to Qualify Ability to DestroyClostridium Test Protein

One purpose of developing the synthetic defined Clostridium surrogateprotein is to provide a method for rapidly determining the effectivenessof sterilization, deimmunization, and/or disinfection by a device, suchas sterilization device, deimmunization device, or disinfection device.In this example, the method for rapidly determining effectivesterilization, deimmunization, and/or disinfection includes multiplesteps including at least: 1) preparing the synthetic defined Clostridiumsurrogate protein test samples, 2) subjecting the defined Clostridiumsurrogate protein test samples to sterilization, deimmunization, ordisinfection, and 3) using Western Blot or similar type analysis tovisualize the effects of sterilization, deimmunization, or disinfectionof defined Clostridium surrogate protein test samples. Successfulsterilization, deimmunization, or disinfection has occurred when all thedefined Clostridium surrogate protein test samples are fragmented and asa result of the protein fragmentation, none remains to bind to thevisualization antibodies indicating the defined Clostridium surrogateprotein was destroyed. If sterilization, deimmunization, or disinfectionwas not successful, protein bands will be seen on the Western Blotanalysis.

Following the process more fully described for the Prion test, a similarprocess was followed to create the Clostridium sterilization,deimmunization, and/or disinfection test. First, to create the samplefor the test using the defined Clostridium surrogate protein, DNAencoding the amino acid SEQ ID NO: 1 for Clostridium above wassynthesized and cloned into standard vectors both for E. coli and yeastexpression. Using standard techniques, large quantities of protein wereproduced in E. coli or yeast and isolated by standard recombinantmethods. Using a nickel column, a full-length defined Clostridiumsurrogate protein (171 amino acids long) was isolated. To create thesamples having the defined Clostridium surrogate protein to qualifysterilization, deimmunization, or disinfection of the samples were driedonto small filter papers, dried inside small tubes or a surface of anobject subjected to sterilization, deimmunization, or disinfection e.g.,using cycles of applying a solvent and microwave energy as disclosed inthe '469 patent application.

After sterilization, deimmunization, or disinfection, the treatedClostridium samples were transferred to tubes, denatured and separatedby size and transferred to nylon membrane before being permanentlycross-linked to the membrane. The final steps include incubating thenylon membrane with a primary antibody that specifically binds to thedefined Clostridium surrogate protein of interest. In the experimentsdiscussed herein, the primary anti-suf I loci encoded protein antibodywas a rabbit polyclonal antibody that was raised against a syntheticpeptide discussed above for SEQ ID NO: 1 residues. For addedsensitivity, addition antibodies, both monoclonal and polyclonal, wereraised against the other synthetic peptide(s). For the Western BlotAnalysis, a secondary antibody may be a HRP-labelled goat anti-rabbit toenable visualization of the protein fragments, both intact andfragmented. If the defined Clostridium surrogate proteins havecompletely fragmented no bands will be visualize on the Western blot.Very small fragments and amino acids will be too small to be retained onthe gel. When successful sterilization, deimmunization, or disinfectionoccurred, the visualized Western blot has a dark ban in the untreatedcontrol sample, e.g., indicated at 210, FIG. 8, and a complete absenceof any bands for the defined Clostridium surrogate protein samplesubjected to sterilization, deimmunization, or disinfection indicatessuccessful sterilization, deimmunization, or disinfection, e.g.,indicated at 212.

Example 8: Comparing Amino Acid Sequence of Multiple Members of theBacillus Genus

It is important to quality the ability of a sterilization device, adeimmunization device or disinfection device to destroy bacteria of anyBacillus species that may be contaminating medical equipment orsupplies. In this example, a defined quantity of the defined Bacillussurrogate protein having the predetermined SEQ ID NO: 2 discussed aboveis subjected to sterilization, deimmunization, or disinfection torapidly determine the effectiveness of the sterilization,deimmunization, or disinfection using Western Blot analysis, proteinarray analysis, or similar type analysis, and the antibody for thedefined Bacillus surrogate protein shown above. In this example, toprotect the human operators of the test, the defined surrogate Bacillussurrogate protein needs to incorporate critical characteristics ofBacillus proteins that are critical for the survival and growth ofmembers of the Bacillus genus while avoiding organisms that can infecthumans.

To design the synthetic defined surrogate Bacillus protein having SEQ IDNO: 2, a protein analysis was conducted comparing the amino acidsequences of the suf I loci gene from multiple species of the Bacillusgenus shown in Table 2 above. In this example, the Sequence IDs (foundin Pubmed, www.ncbi.nlm.nih.gov/Pubmed/) for the suf I loci encodedproteins, multiple Bacillus species, used for the comparative are shownin Table 6 below.

TABLE 6 (Bacillus). Sequences used to determine regions of high homologyin suf 1 locus of multiple Bacillus Species. Species Sequence IDBacillus subtilis AAB62305.1 Bacillus atrophaeus WP_011948579.1 Bacilluspumilus WP_039217212.1

The protein produced by the suf I loci is fundamental to survival andgrowth of a wide range of spores, bacteria and fungus. In species of theBacillus genus, the protein product of the suf I loci is called cotA andis critical for many live stages including strongly contributing to thestability of the spore coat.

To design the synthetic defined Bacillus surrogate protein for SEQ IDNO: 2, the specific amino acids from the proteins listed in Table 6 werealigned to determine amino acid sequence regions that are highlyhomologous in all evaluated Bacillus species as shown in FIG. 9. Theprotein encoded by the suf I loci includes three cupredoxin domains,indicated at 214 for domain 1, 216 for domain 2, and 218 for domain 3.In the Bacillus genus, domain 2, indicated at 216, shows high homologybetween Bacillus species, so SEQ ID NO: 2 above was used as thesynthetic defined Bacillus surrogate protein to be used for the Bacillustest and the corresponding peptide discussed above was used to raise apolyclonal antibody for use by Western Blot analysis. Additionalpolyclonal and monoclonal antibodies were created as needed.

Example 9: Developing Western Test to Qualify Ability to DestroyBacillus Test Protein

One purpose of developing the synthetic defined Bacillus surrogateprotein is to provide a method for rapidly determining the effectivenessof sterilization, deimmunization, and/or disinfection by a device, suchas a sterilization device, deimmunization device, or disinfectiondevice. The method for rapidly determining effective sterilization,deimmunization, and/or disinfection preferably includes multiple stepsincluding at least: 1) preparing synthetic defined Bacillus surrogateprotein test samples, 2) subjecting the Bacillus surrogate protein testsamples to sterilization, deimmunization, or disinfection, and 3) usingWestern Blot or similar analysis to visualize the effects ofsterilization, deimmunization, or disinfection of defined Bacillussurrogate protein test samples. Successful sterilization,deimmunization, or disinfection has occurred when all the definedBacillus surrogate protein test samples are fragmented and as a resultof the protein fragmentation, none remains to bind to the visualizationantibodies indicating the defined Bacillus surrogate protein wasdestroyed. If sterilization, deimmunization, or disinfection was notsuccessful, protein bands will be see on the Western Blot analysis.

Following the process more fully described for the Prion test, a similarprocess was followed to create the Bacillus sterilization,deimmunization, and/or disinfection test. First, to create the samplefor the test using the synthetic defined Bacillus surrogate protein, DNAencoding the amino acid for SEQ ID NO: 2 above was synthesized andcloned into standard vectors both for E. coli and yeast expression.Using standard techniques, large quantities of the defined Bacillussurrogate protein were produced in E. coli or yeast and isolated bystandard recombinant methods. Using a nickel column, a full-lengthdefined Bacillus surrogate protein (171 amino acids long) was isolated.To create the samples having the synthetic defined Bacillus surrogateprotein to qualify sterilization, deimmunization, and/or disinfectionthe samples were dried onto small filter papers, dried inside smalltubes or a surface of an object subjected to sterilization,deimmunization, or disinfection, e.g., using cycles of applying asolvent and microwave energy, e.g., as disclosed in the '469 patentapplication.

After sterilization deimmunization and/or disinfection, the treatedBacillus samples were transferred to tubes, denatured and separated bysize and transferred to nylon membrane before being permanentlycross-linked to the membrane. The final steps include incubating thenylon membrane with a primary antibody that specifically binds to thedefined Bacillus surrogate proteins. In the experiments discussedherein, the primary anti-suf I loci protein antibody was a rabbitpolyclonal antibody that was raised against a synthetic peptidediscussed above for SEQ ID NO: 2 residues. For added sensitivity,addition antibodies, both monoclonal and polyclonal, were raised againstthe other synthetic peptide(s). For the Western Blot Analysis, asecondary antibody may be a HRP-labelled goat anti-rabbit to enablevisualization of the protein fragments, both intact and fragmented. Ifthe defined Bacillus surrogate proteins have completely fragmented nobands will be visualize on the Western blot. Very small fragments andamino acids will be too small to be retained on the gel. When successfulsterilization, deimmunization, or disinfection occurred, the visualizedWestern blot has a dark ban in the untreated control sample, e.g.,indicated at 220, FIG. 10, and a complete absence of any bands fordefined Bacillus surrogate protein sample subjected to sterilization,deimmunization, or disinfection indicates successful sterilization,deimmunization, or disinfection, indicated at 222.

Example 10: Comparing Amino Acid Sequence of Multiple Members of theMycobacterium Genus

It is important to qualify the ability of a sterilization device, adeimmunization device, or a disinfection device to destroy bacteria ofany Mycobacterium species that may be contaminating medical equipment orsupplies. In this example, a defined quantity of the defined surrogateprotein having a SEQ ID NO: 3 discussed above is subjected tosterilization, deimmunization, or disinfection to rapidly determine theeffectiveness of the sterilization, deimmunization, or disinfectionusing Western Blot analysis, protein array analysis, or similar typeanalysis and using an antibody specific for the protein. In thisexample, to protect the human operators of the test, the definedMycobacterium surrogate protein needs to incorporate criticalcharacteristics of Mycobacterium proteins that are critical for thesurvival and growth of members of the Mycobacterium genus while avoidingorganisms that can infect humans.

To design the synthetic defined Mycobacterium surrogate protein SEQ IDNO: 3 above, a protein analysis was conducted comparing the amino acidsequences of the suf I loci gene from multiple species of theMycobacterium genus shown in Table 2 above. In this example, theSequence IDs (found in Pubmed, www.ncbi.nlm.nih.gov/Pubmed/) for the sufI loci proteins, multiple Mycobacterium species, used for thecomparative are shown in Table 7 below:

TABLE 7 (Mycobacteria). Sequences used to determine regions of highhomology in suf 1 locus of multiple Mycobacterium Species. SpeciesSequence ID Mycobacterium tuberculosis WP_003404392.1 Mycobacteriumafricanum KBG17039.1 Mycobacterium kansasii WP_023367763.1

The protein produced by the suf I loci is fundamental to survival andgrowth of a wide range of spores, bacteria and fungus. In species of theMycobacterium genus, the protein product of the suf I loci is calledcumA and is critical for many live stages including cell survival andgrowth.

To design the synthetic defined Mycobacterium surrogate protein havingSEQ ID NO: 3, the specific amino acids from the proteins listed in Table7 were aligned to determine amino acid sequence regions that are highlyhomologous in all evaluated Mycobacterium species as shown in FIG. 11.The protein encoded by the suf I loci includes three cupredoxin domainsthat are indicated at 230 for domain 1, 232 for domain 2, and 234 fordomain 3. In the Mycobacterium genus, domain 2, indicated at 232, showshigh homology between Mycobacterium species so SEQ ID NO: 3 above wasused to for the design for synthetic defined Mycobacterium surrogateprotein to be created for the Mycobacterium test and the correspondingpeptide for the defined Mycobacterium surrogate protein discussed abovewas used for a polyclonal antibody for use by Western Blot analysis.

Example 11 Developing Western Test to Qualify Ability to DestroyMycobacterium Test Protein

One purpose of developing the defined Mycobacterium surrogate protein isto provide a method for rapidly determining the effectiveness ofsterilization, deimmunization, and/or disinfection by a device, such asa sterilization device, a deimmunization device, or a disinfectiondevice. The method for rapidly determining effective sterilization,deimmunization, and/or disinfection preferably includes multiple stepsincluding at least: 1) preparing the defined Mycobacterium surrogateprotein test samples, 2) subjecting the defined Mycobacterium surrogateprotein test samples to sterilization, deimmunization, or disinfection,and 3) using Western Blot or similar analysis to visualize the effectsof sterilization, deimmunization, and/or disinfection of syntheticdefined Mycobacterium surrogate protein test samples. Successfulsterilization, deimmunization, and/or disinfection has occurred when allthe synthetic defined Mycobacterium surrogate protein test samples arefragmented and as a result of the protein fragmentation, none remains tobind to the visualization antibodies indicating the definedMycobacterium protein was destroyed. If sterilization, deimmunization,and/or disinfection was not successful, protein bands will be see on theWestern Blot analysis.

Following the process more fully described for the Prion test, a similarprocess was followed to create the Mycobacterium sterilization,deimmunization, and/or disinfection test. First, to create the samplefor the test using the defined Mycobacterium surrogate protein, DNAencoding the amino acid SEQ ID NO: 3 above was synthesized and clonedinto standard vectors both for E. coli and yeast expression. Usingstandard techniques, large quantities of protein were produced in E.coli or yeast and isolated by standard recombinant methods. Using anickel column, a full-length synthetic Mycobacterium surrogate protein(171 amino acids long) was isolated. To create the samples having theMycobacterium surrogate protein to qualify sterilization,deimmunization, and/or disinfection the samples were dried onto smallfilter papers, dried inside small tubes or a surface of an objectsubjected to sterilization, deimmunization, and/or disinfection, e.g.,using cycles of applying a solvent and microwave energy as disclosed inthe '469 patent application.

After sterilization, deimmunization, and/or disinfection the treatedMycobacterium surrogate protein samples were transferred to tubes,denatured and separated by size and transferred to nylon membrane beforebeing permanently cross-linked to the membrane. The final steps includeincubating the nylon membrane with a primary antibody that specificallybinds to the protein of interest. In the experiments discussed herein,the primary anti-suf I loci protein antibody was a rabbit polyclonalantibody that was raised against a synthetic peptide discussed above forSEQ ID NO: 3 for Mycobacterium above residues. For added sensitivity,addition antibodies, both monoclonal and polyclonal, were raised againstthe other synthetic peptide(s). For the Western Blot Analysis, asecondary antibody may be a HRP-labelled goat anti-rabbit to enablevisualization of the protein fragments, both intact and fragmented. Ifthe Mycobacterium surrogate proteins have completely fragmented no bandswill be visualize on the Western blot. Very small fragments and aminoacids will be too small to be retained on the gel. When successfulsterilization, deimmunization, and/or disinfection has occurred, thevisualized Western blot has a dark ban in the untreated control sample,e.g., indicated at 236, FIG. 12, and a complete absence of any bands forthe Mycobacterium surrogate protein sample subjected to sterilization,deimmunization, or disinfection indicates successful sterilization,deimmunization, or disinfection, e.g., indicated at 238.

Example 12: Comparing Amino Acid Sequence of Multiple Members of theStaphylococcus Genus

It is important to qualify the ability of a sterilization device, adeimmunization device, or a disinfection device to destroy bacteria ofany Staphylococcus species that may be contaminating medical equipmentor supplies. In this example, a defined quantity of the definedStaphylococcus surrogate protein having the predetermined SEQ ID NO: 4discussed above is subjected to sterilization, deimmunization, ordisinfection to rapidly determine the effectiveness of thesterilization, deimmunization, or disinfection using Western Blotanalysis, protein array analysis, or similar type analysis and theantibody specific for the defined Staphylococcus surrogate protein. Inthis example, to protect the human operators of the test, the syntheticdefined Staphylococcus surrogate protein needs to incorporate criticalcharacteristics of Staphylococcus proteins that are critical for thesurvival and growth of members of the Staphylococcus genus whileavoiding organisms that can infect humans.

To design the synthetic defined Staphylococcus surrogate protein havingSEQ ID NO: 4, a protein analysis was conducted comparing the amino acidsequences of the suf I loci gene from multiple species of theStaphylococcus genus shown in Table 2 above. In this example, theSequence IDs (found in Pubmed, www.ncbi.nlm.nih.gov/Pubmed/) for the sufI loci proteins, multiple Staphylococcus species, used for thecomparative are shown in Table 8 below:

TABLE 8 (Staphylucoccus). Sequences used to determine regions of highhomology in Suf 1 locus of multiple Staphylucoccus Species. SpeciesSequence ID Staphylucoccus aureus WP_000282432.1 Staphylucoccusepidermidis WP_023567454.1 Staphylucoccus saprophyticus OEK13316.1

The protein produced by the suf I loci is fundamental to survival andgrowth of a wide range of spores, bacteria and fungus. In species of theStaphylococcus genus, the protein product of the suf I loci is calledcueO and is critical for many live stages including stronglycontributing to cell survival and growth

To design the synthetic defined Staphylococcus surrogate protein, thespecific amino acids from the proteins listed in Table 8 were aligned todetermine amino acid sequence regions that are highly homologous in allevaluated Staphylococcus species as shown in FIG. 13. The proteinencoded by the suf I loci includes three cupredoxin domains that areindicated at 240 for domain 1, 242 for domain 2, and 244 for domain 3.In the Staphylococcus genus, domain 2 indicated at 242 shows highhomology between Staphylococcus species, so SEQ ID NO: 4 above was usedto for the design of the synthetic defined Staphylococcus surrogateprotein to be created for the Staphylococcus test and the correspondingpeptide discussed above was used for a polyclonal antibody for use byWestern Blot analysis.

Example 13: Developing Western Test to Qualify Ability to DestroyStaphylococcus Test Protein

One purpose of developing the defined Staphylococcus surrogate proteinis to provide a method for rapidly determining the effectiveness ofsterilization, deimmunization, and/or disinfection device, such assterilization device, deimmunization device, or disinfection deviceand/or supplies. The method for rapidly determining effectivesterilization, deimmunization, and/or disinfection includes multiplesteps including at least: 1) preparing synthetic defined Staphylococcussurrogate protein test samples, 2) subjecting the defined Staphylococcussurrogate protein test samples to sterilization, deimmunization, and/ordisinfection, and 3) using Western Blot or similar analysis to visualizethe effects of sterilization, deimmunization, and/or disinfection ofdefined Staphylococcus surrogate protein test samples of the definedsurrogate protein. Successful sterilization has occurred when all thedefined Staphylococcus surrogate protein test samples are fragmented andas a result of the protein fragmentation, none remains to bind to thevisualization antibodies indicating the defined Staphylococcus surrogateprotein was destroyed. If sterilization, deimmunization, and/ordisinfection was not successful, protein bands will be see on theWestern Blot analysis.

Following the process more fully described for the Prion test, a similarprocess was followed to create the Staphylococcus sterilization,deimmunization, or disinfection test. First to create the sample for thetest using the synthetic defined Staphylococcus surrogate protein, DNAencoding the amino acid SEQ ID NO: 4 above was synthesized and clonedinto standard vectors both for E. coli and yeast expression. Usingstandard techniques, large quantities of the defined Staphylococcussurrogate protein were produced in E. coli or yeast and isolated bystandard recombinant methods. Using a nickel column, a full-lengthdefined Staphylococcus surrogate protein (171 amino acids long) wasisolated. To create the samples having the synthetic definedStaphylococcus surrogate protein to qualify sterilization,deimmunization, and/or disinfection the samples were dried onto smallfilter papers, dried inside small tubes or a surface of an objectsubjected to sterilization, deimmunization, and/or disinfection e.g.,using cycles of applying a solvent and microwave energy as disclosed inthe '469 patent application.

After sterilization, deimmunization, and/or disinfection, the treateddefined Staphylococcus surrogate protein samples were transferred totubes, denatured and separated by size and transferred to nylon membranebefore being permanently cross-linked to the membrane. The final stepsinclude incubating the nylon membrane with a primary antibody thatspecifically binds to the Staphylococcus surrogate protein of interest.In the experiments discussed herein, the primary anti-suf I loci proteinantibody was a rabbit polyclonal antibody that was raised against asynthetic peptide discussed above for SEQ ID NO: 4 residues. For addedsensitivity, addition antibodies, both monoclonal and polyclonal, wereraised against the other synthetic peptide(s). For the Western Blotanalysis, a secondary antibody may be a HRP-labelled goat anti-rabbit toenable visualization of the protein fragments, both intact andfragmented. If the defined Staphylococcus surrogate proteins havecompletely fragmented no bands will be visualize on the Western blot.Very small fragments and amino acids will be too small to be retained onthe gel. When successful sterilization, deimmunization, and/ordisinfection occurred, the visualized Western blot has a dark ban in theuntreated control sample, indicated at 250, FIG. 14, and a completeabsence of any bands for defined Staphylococcus surrogate protein samplesubjected to sterilization, deimmunization, or disinfection indicatessuccessful sterilization, deimmunization, or disinfection, indicated at252.

Example 14: Comparing Amino Acid Sequence of Multiple Members of thePseudomonas Genus

It is important to qualify the ability of a sterilization device, adeimmunization device or disinfection device to destroy bacteria of anyPseudomonas species that may be contaminating medical equipment orsupplies. In this example, a defined Pseudomonas surrogate proteinquantity of the defined surrogate protein having a predetermined SEQ IDNO: 5 discussed above is subjected to sterilization, deimmunization, ordisinfection to rapidly determine the effectiveness of thesterilization, deimmunization, or disinfection using Western Blotanalysis, protein array analysis, or similar type analysis, and using anantibody specific for the defined Pseudomonas surrogate protein shownabove. In this example, to protect the human operators of the test, thedefined Pseudomonas surrogate protein needs to incorporate criticalcharacteristics of Pseudomonas proteins that are critical for thesurvival and growth of members of the Pseudomonas genus while avoidingorganisms that can infect humans.

To design the synthetic defined Pseudomonas surrogate protein having SEQID NO: 5, a protein analysis was conducted comparing the amino acidsequences of the suf I loci gene from multiple species of thePseudomonas genus shown in Table 2 above. In this example, the SequenceIDs (found in Pubmed, www.ncbi.nlm.nih.gov/Pubmed/) for the suf I lociTable 9 below:

TABLE 9 Pseudomonas. Sequences used to determine regions of highhomology in suf 1 locus of multiple Pseudomonas Species. SpeciesSequence ID Pseudomonas aeruginosa WP_023096478.1 Pseudomonasfluorescens WP_003227851.1 Pseudomonas putida WP_019750583.1

The protein produced by the suf I loci is fundamental to survival andgrowth of a wide range of spores, bacteria and fungus. In species of thePseudomonas genus, the protein product of the suf I loci is called cumAand is critical for many live stages including strongly contributing tothe cell survival and growth

To design the defined Pseudomonas surrogate protein, the specific aminoacids front the proteins listed in Table 9 were aligned to determineamino acid sequence regions that are highly homologous in all evaluatedPseudomonas species as shown in FIG. 15. The protein encoded by the sufI loci includes three cupredoxin domains indicated at 260 for domain 1,262 for domain 2, and 264 for domain 3. In the Pseudomonas genus, domain2, indicated at 262, shows high homology between Pseudomonas species soSEQ ID NO: 5 above was used to for she design of the synthetic surrogateprotein to be created for the defined Pseudomonas surrogate protein testand the corresponding peptide shown above was used for a polyclonalantibody for use by Western Blot analysis.

Example 15: Developing Western Test to Qualify Ability to DestroyPseudomonas Test Protein

One purpose of developing the defined Pseudomonas surrogate protein testis to create a method for rapidly determining the effectiveness ofsterilization, deimmunization, and/or disinfection by a device, such asa sterilization device, a deimmunization device, or a disinfectiondevice. The method for rapidly determining effective sterilization,deimmunization, and/or disinfection preferably includes multiple stepsincluding at least: 1) preparing synthetic defined Pseudomonas surrogateprotein test samples, 2) subjecting the synthetic Pseudomonas surrogateprotein test samples to sterilization, deimmunization, and/ordisinfection, and 3) using Western Blot or similar analysis to visualizethe effects of sterilization, deimmunization, and/or disinfection ofsynthetic defined Pseudomonas surrogate test samples. Successfulsterilization has occurred when all the defined Pseudomonas surrogateprotein test samples are fragmented and as a result of the proteinfragmentation, none remains to bind to the visualization antibodiesindicating the defined Pseudomonas surrogate protein was destroyed. Ifsterilization, deimmunization, and/or disinfection was not successful,protein bands will be see on the Western Blot analysis.

Following the process more fully described for the Prion test, a similarprocess was followed to create the defined Pseudomonas sterilization,deimmunization and/or disinfection test. First, to create the sample forthe test using the defined Pseudomonas surrogate protein, DNA encodingthe amino acid SEQ ID NO: 5 defined above was synthesized and clonedinto standard vectors both for E. coli and yeast expression. Usingstandard techniques, large quantities of the defined Pseudomonassurrogate protein were produced in E. coli or yeast and isolated bystandard recombinant methods. Using a nickel column, a full-lengthsynthetic defined Pseudomonas surrogate protein (171 amino acids long)was isolated. To create the samples having the synthetic definedsurrogate protein to qualify sterilization, deimmunization, and/ordisinfection of the samples were dried onto small filter papers, driedinside small tubes or a surface of an object subjected to sterilization,deimmunization, and/or disinfection, e.g., using cycles of applying asolvent and microwave energy as disclosed in the '469 patentapplication.

After sterilization, deimmunization, and/or disinfection, the treateddefined Pseudomonas surrogate protein samples were transferred to tubes,denatured and separated by size and transferred to nylon membrane beforebeing permanently cross-linked to the membrane. The final steps includeincubating the nylon membrane with a primary antibody that specificallybinds to the defined Pseudomonas surrogate protein. In the experimentsdiscussed herein, the primary anti-suf I loci protein antibody was arabbit polyclonal antibody that was raised against a synthetic peptidediscussed above for SEQ ID NO: 5 residues. For added sensitivity,addition antibodies, both monoclonal and polyclonal, were raised againstthe other synthetic peptide(s). For the Western Blot Analysis, asecondary antibody may be a MRP-labelled goat anti-rabbit to enablevisualization of the protein fragments, both intact and fragmented. Ifthe synthetic defined Pseudomonas surrogate proteins have completelyfragmented no bands will be visualize on the Western blot. Very smallfragments and amino acids will be too small to be retained on the gel.When successful sterilization, deimmunization, or disinfection occurred,the visualized Western blot has a dark ban in the untreated controlsample, e.g., indicated at 266, FIG. 16, and a complete absence of anybands for the defined Pseudomonas surrogate protein sample subjected tosterilization, deimmunization, or disinfection indicates successfulsterilization, deimmunization, or disinfection, indicated at 268.

Example 16: Comparing Amino Acid Sequence of Multiple Members of theTrichophyton Genus

It is important to qualify the ability of a sterilization device, adeimmunization device, and/or a disinfection device to destroy bacteriaof any Trichophyton species that may be contaminating medical equipment.In this example, a defined quantity of the defined Trichophytonsurrogate protein having a predetermined SEQ ID NO: 6 discussed above issubjected to sterilization, deimmunization, or disinfection to rapidlydetermine the effectiveness of the sterilization, deimmunization, ordisinfection using Western Blot analysis, protein array analysis, orsimilar type analysis and using the antibody specific for the definedTrichophyton surrogate protein. In this example, to protect the humanoperators of the test, the synthetic defined Trichophyton surrogateprotein needs to incorporate critical characteristics of Trichophytonproteins that are critical for the survival and growth of members of theTrichophyton genus while avoiding organisms that can infect humans.

To design the synthetic defined Trichophyton surrogate protein havingthe SEQ ID NO: 6, a protein analysis was conducted comparing the aminoacid sequences of the suf I loci gene from multiple species of theTrichophyton genus shown in Table 2 above. In this example, the SequenceIDs (found in Pubmed, www.ncbi.nlm.nih.gov/Pubmed/) for the suf I lociproteins, multiple Trichophyton species, used for the comparative areshown in Table 10 below:

TABLE 10 Trichophyton. Sequences used to determine regions of highhomology in suf 1 locus of Trichophyton Species. Species Sequence IDTrichophyton rubrum XP_003236812.1 Trichophyton tonsurane EGD95875.1

The protein produced by the suf I loci is fundamental to survival andgrowth of a wide range of spores, bacteria and fungus. In species of theTrichophyton genus, the protein product of the suf I loci is calledlaccase and is critical for many live stages including stronglycontributing to cell survival and growth

To design the synthetic defined Trichophyton surrogate protein, thespecific amino acids from the proteins listed in Table 10 above werealigned to determine amino acid sequence regions that are highlyhomologous in all evaluated Trichophyton species as shown in FIG. 17.The protein encoded by the suf I loci includes three cupredoxin domainsthat are indicated at 270 for domain 1, 272 for domain 2, and 274 fordomain 3. In the Trichophyton genus, domain 2, indicated at 272, showshigh homology between Trichophyton species so SEQ ID NO: 6 above wasused to for the design of the synthetic defined Trichophyton surrogateprotein to be created for the Trichophyton test and the correspondingpeptide discussed above was used for a polyclonal antibody for use byWestern Blot analysts.

Example 17: Developing Western Test to Qualify Ability to DestroyTrichophyton Test Protein

One purpose of developing the defined Trichophyton surrogate protein isto provide a method for rapidly determining the effectiveness ofsterilization, deimmunization, and/or disinfection by a device, such assterilization device, a deimmunization device, or a disinfection device.The method for rapidly determining effective sterilization,deimmunization, or disinfection preferably includes multiple stepsincluding at least: 1) preparing synthetic defined Trichophytonsurrogate protein test samples, 2) subjecting the defined Trichophytonsurrogate protein test samples to sterilization, deimmunization, ordisinfection, and 3) using Western Blot or similar analysis to visualizethe effects of sterilization, deimmunization, or disinfection of definedTrichophyton surrogate protein test samples. Successful sterilization,deimmunization, or disinfection has occurred when all the definedTrichophyton surrogate protein test samples are fragmented and as aresult of the protein fragmentation, none remains to bind to thevisualization antibodies indicating the defined Trichophyton surrogateprotein was destroyed. If sterilization, deimmunization and/ordisinfection was not successful, protein bands will be see on theWestern Blot analysis.

Following the process more fully described for the Prion test, a similarprocess was followed to create the Trichophyton sterilization,deimmunization and/or disinfection test. First, to create the sample forthe test using the synthetic defined Trichophyton surrogate protein, DNAencoding the amino acid SEQ ID NO: 7 above was synthesized and clonedinto standard vectors both for E. coli and yeast expression. Usingstandard techniques, large quantities of defined Trichophyton surrogateprotein were produced in E. coli or yeast and isolated by standardrecombinant methods. Using a nickel column, a full-length syntheticdefined Trichophyton surrogate protein (171 amino acids long) wasisolated. To create the samples having the synthetic definedTrichophyton surrogate protein to qualify sterilization, deimmunization,or disinfection, the defined Trichophyton surrogate protein samples weredried onto small filter papers, dried inside small tubes or a surface ofan object subjected to sterilization, deimmunization and/ordisinfection, e.g., using cycles of applying a solvent and microwaveenergy as disclosed in the '469 patent application.

After sterilization, deimmunization, or disinfection, the treatedTrichophyton samples were transferred to tubes, denatured and separatedby size and transferred to nylon membrane before being permanentlycross-linked to the membrane. The final steps include incubating thenylon membrane with a primary antibody that specifically binds to theprotein of interest. In the experiments discussed herein, the primaryanti-suf I loci protein antibody was a rabbit polyclonal antibody thatwas raised against a synthetic peptide discussed above for SEQ ID NO: 6residues. For added sensitivity, addition antibodies, both monoclonaland polyclonal, were raised against the other synthetic peptide(s). Forthe Western Blot analysis, a secondary antibody may be a HRP-labelledgoat anti-rabbit to enable visualization of the protein fragments, bothintact and fragmented. If the proteins have completely fragmented nobands will be visualize on the Western blot. Very small fragments andamino acids will be too small to be retained on the gel. When successfulsterilization, deimmunization, or disinfection has occurred, thevisualized Western blot has a dark ban in the untreated control sample,e.g., indicated at 276, FIG. 18, and a complete absence of any bands forthe defined Trichophyton surrogate protein sample subjected tosterilization, deimmunization, or disinfection indicates successfulsterilization, deimmunization, or disinfection, indicated at 278.

Example 18: Comparing Amino Acid Sequence of Multiple Members of theCandida Genus

It is important to qualify the ability of a sterilization device, adeimmunization device, or disinfection device, to destroy bacteria ofany Candida species that may be contaminating medical equipment. In thisexample, a defined quantity of the defined Candida surrogate proteinhaving a predetermined SEQ ID NO: 7 discussed above is subjected tosterilization, deimmunization, or disinfection to rapidly determine theeffectiveness of the sterilization, deimmunization, or disinfectionusing Western Blot analysis, protein array analysis, or similar typeanalysis and the antibody specific for the defined Candida surrogateprotein. In this example, to protect the human operators of the test,the defined Candida surrogate protein needs to incorporate criticalcharacteristics of Candida proteins that are critical for the survivaland growth of members of the Candida genus while avoiding organisms thatcan infect humans.

To design the synthetic defined Candida surrogate protein having the SEQID NO: 7, a protein analysis was conducted comparing the amino acidsequences of the suf I loci gene from multiple species of the Candidagenus shown in Table 2 above. In this example, the Sequence IDs (foundin Pubmed, www.ncbi.nlm.nih.gov/Pubmed/) for the suf I loci proteins,multiple Candida species, used for the comparative are shown in Table 11below:

TABLE 11 (Candida). Sequences used to determine regions of high homologyin Suf 1 locus of Candida Species. Species Sequence ID Candida albicansKHC71512.1 Candida dubliniensis XP_002420841.1 Candida tropicalisXP_002548698.1 Candida auris XP_018169615.1

The protein produced by the suf I loci is fundamental to survival andgrowth of a wide range of spores, bacteria and fungus. In species of theCandida genus, the protein product of the suf I loci is called laccaseand is critical for many live stages including strongly contributing tocell survival and growth

To design the synthetic defined Candida surrogate protein, the specificamino acids from the proteins listed in Table 11 were aligned todetermine amino acid sequence regions that are highly homologous in allevaluated Candida species as shown in FIG. 19. The protein encoded bythe suf I loci includes three cupredoxin domains, indicated at 280 fordomain 1, 282 for domain 2, and 284 for domain 3. In the Candida genus,domain 2, indicated at 284, shows high homology between Candida speciesso SEQ ID NO: 7 above was used to for the design of the syntheticdefined Candida surrogate protein to be created for the Candida test andthe corresponding peptide discussed above was used for a polyclonalantibody for use by Western Blot analysis.

Example 19: Developing Western Test to Qualify Ability to DestroyCandida Test Protein

One purpose of developing the defined Candida surrogate protein test isto provide a method for rapidly determining the effectiveness ofsterilization, deimmunization, and/or disinfection by a device, such asa sterilization device, a deimmunization device, or a disinfectiondevice. The method for rapidly determining effective sterilization,deimmunization, and/or disinfection includes multiple steps preferablyincluding at least: 1) preparing synthetic defined Candida surrogateprotein test samples, 2) subjecting the defined Candida surrogateprotein test samples to sterilization, deimmunization, or disinfection,and 3) using Western Blot or similar analysis to visualize the effectsof sterilization, deimmunizations or disinfection of defined Candidasurrogate protein test samples. Successful sterilization,deimmunization, or disinfection has occurred when ail the definedCandida surrogate protein test samples are fragmented and as a result ofthe protein fragmentation, none remains to bind to the visualizationantibodies indicating the defined Candida surrogate protein wasdestroyed. If sterilization, deimmunization, or disinfection was notsuccessful, protein bands will be see on the Western Blot analysis.

Following the process more folly described for the Prion test, a similarprocess was followed to create the defined Candida surrogate proteinsterilization, deimmunization and/or disinfection test. First, to createthe sample for the synthetic defined Candida surrogate protein testusing the defined Candida surrogate protein, DNA encoding the amino acidSEQ ID NO: 7 was synthesized and cloned into standard vectors both forE. coli and yeast expression. Using standard techniques, largequantities of protein were produced in E. coli or yeast and isolated bystandard recombinant methods. Using a nickel column, a full-lengthsynthetic defined Candida surrogate protein (171 amino acids long) wasisolated. To create the samples having the synthetic defined Candidasurrogate protein to qualify sterilization, deimmunization, ordisinfection, the samples having the defined Candida surrogate proteinwere dried onto small filter papers, dried inside small tubes or asurface of an object subjected to sterilization, deimmunization, and/ordisinfection, e.g., using cycles of applying a solvent and microwaveenergy as discussed in the '469 patent application.

After sterilization, deimmunization, or disinfection, the treateddefined Candida surrogate protein samples were transferred to tubes,denatured and separated by size and transferred to nylon membrane beforebeing permanently cross-linked to the membrane. The final steps includeincubating the nylon membrane with a primary antibody that specificallybinds to the defined Candida surrogate protein. In the experimentsdiscussed herein, the primary anti-suf I loci protein antibody was arabbit polyclonal antibody that was raised against a synthetic peptidediscussed above for SEQ ID NO: 7 residues. For added sensitivity,addition antibodies, both monoclonal and polyclonal, were raised againstthe other synthetic peptide(s). For the Western Blot analysis, asecondary antibody may be a HRP-labelled goat anti-rabbit to enablevisualization of the protein fragments, both intact and fragmented. Ifthe proteins have completely fragmented no bands will be visualize onthe Western blot. Very small fragments and amino acids will be too smallto be retained on the gel. When successful sterilization,deimmunization, or disinfection has occurred, the visualized Westernblot has a dark ban in the untreated control sample, indicated at 286,FIG. 20, and a complete absence of any bands for the defined Candidasurrogate protein sample subjected to sterilization, deimmunization, ordisinfection indicates successful sterilization, deimmunization, ordisinfection, indicated at 290.

Example 20: Immobilizing Proteins Onto a Solid Surface

Many processes and solid substrates are well known in the art forimmobilizing proteins onto a solid surface. In this example, glassslides were obtained having round wells created by printing the glasswith highly water-repellent mark, e.g., as shown by multi-well glassslides 130, 134, FIG. 2, or multi-well glass slide 152, FIG. 3. In thisexample, the glass slides were washed with acetone and milli-Q waterbefore soaking overnight in 1 M NaOH (room temperature). To create aminosilane-treated slides, washed slides using milli-Q water and 99.5%ethanol, were treated with 3-aminopropyltriethoxysilane for 2 hours atroom temperature, washed with milli-Q water, and baked for 2 hr. at 100°C. The multi-well slides were soaked in 1% (v/v) glutaraldehydeovernight at 37° C. to produce glutaraldehyde-treated slides. The slideswere then incubated overnight at 37° C. in chitosan (0.05% (w/v))dissolved in 0.1 M acetic acid buffer (pH 5.0) supplemented with 0.25 mMsodium azide. The treated slides were rinsed twice with 0.1 M aceticacid buffer (pH 5) and then three times with milli-Q water before beingsoaked in 1% (v/v) glutaraldehyde overnight at 37° C. The slides wereincubated overnight at 37° C. in a 0.05% (w/v) solution ofN-(5-Amino-1-carboxy pentyl) iminodiacetic acid (AB-NTA) in 0.1 M HEPESbuffer (pH 8.0), then rinsed 3 times with milli-Q water. The slides werethen soaked in blocking solution (1% (v/v) glycine) for 1 h at 37° C.,then rinsed 3 times with milli-Q water, 3 times with 0.5 M NiCl2, and 3times with milli-Q water to produce Ni-NTA immobilized slides ready forprotein immobilization.

In this example, the defined Clostridium surrogate protein having SEQ IDNO: 1 was diluted in TG buffer (50 mM Tris-HCl (pH 8.0), 10% (v/v)glycerol) to 200 μg/ml. Different quantities of the defined Clostridiumsurrogate protein, e.g., 200 ng, 100 ng, 50 ng, 25 ng, 12.5 ng, 6.25 ng,3.125 ng, 1.562 ng, 0.781 ng and 0 ng, were spotted in individual wells,e.g., the wells exemplarily indicated at 132 and 136, FIG. 2, andincubated for 60 min in a moist, dark chamber and finished by washingwith TG buffer. After subjecting the slides 130 and 134 tosterilization, deimmunization, or disinfection using cycles of a solventand electromagnetic radiation, e.g., microwaves as disclosed in the '469patent application, slides 130 and 134 were incubated withanti-Clostridium antibodies discussed above raised against the peptideselected from SEQ ID NO: 1. To visualize the amount of the definedClostridium surrogate protein remaining after sterilization,deimmunization, or disinfection, the slides were then treated withanti-Ig antibody labeled with the fluorescence dye fluorescenceisothiocynate (FITC). A matched slide that was not sterilized in thisexample, slide 130, was used as a control for the protein arrayanalysis. Using a fluorescence microscope with camera, the loss ofprotein can be visualized that can be detected with the specificanti-Clostridium antibodies. With additional fluorescence detectionequipment, such as automated readers, increasing sensitivity andtitration of detection can be added. An example of successfulsterilization, deimmunization and/or disinfection is indicated at 140,for slide 134, FIG. 2, when compared against control slide 130 whichvisibly shows color, indicated by the shading in wells 132. Confirmatorytests were conducted by including samples of intact C. sporogenes usingcycles of a solvent and electromagnetic energy or microwaves, e.g., asdisclosed in the '469 patent application. After treatment, the intactbacteria were processed following industry standard protocols. Similarmethods may be used for any of Examples 6 to 18 above.

Example 21: Comparing Amino Acid Sequence of α-Gliadin from AppropriateHuman Consumed Grains

To develop a test to qualify the ability of a sterilization device,deimmunization, or disinfection to destroy medically importantimmunogenic proteins, such as α-gliadin, that may be contaminatingmedical equipment, the following experiments were conducted. In thisexample, to rapidly detect effective deimmunization, a defined qualityof an isolated protein needs to be deimmunized with a deimmunizationdevice, e.g., using cycles of a combination of a solvent andelectromagnetic microwaves radiation, e.g., microwaves, as disclosed inthe '469 patent application and then evaluated by Western Blot analysisusing an antibody specific for the protein. To design a candidateprotein to serve as the representative defined surrogate α-gliadinprotein having a predetermined sequence, the translated sequences ofrepresentative α-gliadin genes from many commonly consumed human grainswere aligned. See Table 13 and Table 14 below. Gliadin protein is theimmunogenic component of gluten and must be avoided by Celiac patients.FIG. 21 shows the amino acid numbering aligned with an example of Breadwheat α-Gliadin. The underline region, indicated at 400, is thepolypeptide that acts as common immunogen in a majority of CeliacPatients. Regions at 402, 404 indicate protein region used to designtest immunogen reagent.

TABLE 13 Sequence ID of α-Gliadin Proteins or Prolamins in Common GrainsUsed to Design Recombinant Protein Sequence. Sequence ID Common NameSpecies Name Number w/Link Bread Wheat Triticum aestivum pir||A27319Common Wheat Triticum sphaerococcum ABQ45316.1 Durum wheat/PastaTriticum turgidum subsp. ADA83698.1 Wheat durum Farro/Emmer WheatTriticum dicoccon AKC91191.1 Macha Wheat Triticum macha AKC91223.1 RyeSecale cereale AFK32718.1 Spelt/Dinkel Wheat Triticum spelta APU92351.1Red Wild Einkor Triticum urartu AKC91171.1 Precursor αGliadin Triticumaestivum Q41545 Bateman et al 2004

TABLE 14 Additional α-Gliadin Protein Sequences that could be used todesign Recombinant Protein. Sequence Species Common ID Species CommonName Sequence ID Triticum Bread pir||A27319 Triticum Durum wheat, pastaADA83698.1 aestivum Wheat P04726.1 turgidum wheat or macaroni ADA83690.1subsp. wheat durum AED99851.1 Triticum Emmer wheat or Farro AKC91191.1dicoccon SCW25751.1 Triticum red wild einkor AKC91171.1 urartuAHY37812.1 Triticum Macha wheat AKC91223.1 macha AHY37818.1 Secale RyeAFK32718.1 cereale AFX69612.1 ABQ52118.1 Aegilops Tausch's goatgrassAFX69602.1 AFX69616.1 tauschii XP_020186089.1 AFX69586.1 AKC91337.1Triticum Common ABQ45316.1 ABQ52112.1 sphaerococcum wheat Triticumspelta Spelt or APU92351.1 Aegilops Sharon goatgrass AMS25611.1 dinkelAPU92675.1 sharonensis AMS25610.1 wheat APU92554.1 AMS25614.1 APU92300.1Aegilops Goatgrass AKC91312.1 searsii APU92425.1 AKC91311.1 APU92357.1Aegilops Goatgrass AHN85624.1 speltoides APU92415.1 Aegilops GoatgrassAHN85626.1 speltoides APU92583.1 Aegilops Goatgrass AEV55370.1uniaristata APU92336.1 Thinopyrum Wild Thinopyrum ADP94197.1 APU92334.1bessarabicum grassesIn this example, the defined α-Gliadin surrogate protein has thefollowing predetermined sequence:For α-Gliadin:

(SEQ ID NO. 8)         10         20         30         40MKTVRVPVPQ PQPQNPSQPQ PQRQVPLVQQ QQFPGQQQQF         50PPQQPYPQPQ PFPSQQPYLQ LQPFPQPQPF PPQLPYHHHH HHThe peptide used for the development of monoclonal or polyclonalantibody used by Western Blot analysis for the above sequence is:

FPPQQPYPQPQPFPSQQPYLQLQPFPQPQ (SEQ ID NO: 24)A fragment of this peptide (KLQPFPQPELPYPQPQ (SEQ ID NO: 25)) in form isthe medically important immunogen is CD.

If the defined α-Gliadin surrogate proteins have completely fragmented,no bands will be visualized on the Western Blot. Very small fragments inamino acid would be too small to be retained on the gel. When successfuldeimmunization, in this example, has occurred, the visualized WesternBlot has a dark band in the untreated control sample, e.g., indicated at420, FIG. 22, and a complete absence of any bands for the definedα-Gliadin surrogate protein sample subject to deimmunization indicatessuccessful deimmunization, e.g., indicated at 422.

Confirmatory tests were conducted by including samples of intact wheatflour using a combination of cycles of a solvent and microwaves, e.g.,as disclosed in the '469 Patent application. After treatment, the intactwheat flour samples were processed following industry standard protocolsto test for the presence of gluten in food stuffs.

Although specific features of the invention are shown in some drawingsand not in others, this is for convenience only as each feature may becombined with any or all of the other features in accordance with theinvention. The words “including”, “comprising”, “having”, and “with” asused herein are to be interpreted broadly and comprehensively and arenot limited to any physical interconnection. Moreover, any embodimentsdisclosed in the subject application are not to be taken as the onlypossible embodiments. Other embodiments will occur to those skilled inthe art and are within the following claims.

In addition, any amendment presented during the prosecution of thepatent application for this patent is not a disclaimer of any claimelement presented in the application as filed: those skilled in the artcannot reasonably be expected to draft a claim that would literallyencompass all possible equivalents, many equivalents will beunforeseeable at the time of the amendment and are beyond a fairinterpretation of what is to be surrendered (if anything), the rationaleunderlying the amendment may bear no more than a tangential relation tomany equivalents, and/or there are many other reasons the applicantcannot be expected to describe certain insubstantial substitutes for anyclaim element amended.

What is claimed is:
 1. A method for determining effective sterilization, deimmunization, and/or disinfection of equipment and/or supplies by a device, the method comprising: providing a defined surrogate protein having a predetermined sequence defined by a sequence which at least about 95% homologous to the sequence (SEQ ID NO. 2)         10         20         30         40 MTLEKTYYEV TMEECTHQLH RDLPPTRLWG YNGLFPGPTI         50 EVKRNENVYV KWMNNLPSTH FLPIDHTIHH SDSQHEEPEV KTVVHLHGGV TPDDSDGYPE AWFSKDFEQT GPYFKREVYH YPNQQRGAIL WYHDHAMALT RLNVYAGLVG AYIIHDPKEK RLKHHHHHH

representative of an infectious agent potentially contaminating the equipment and/or the supplies to be sterilized, deimmunized, and/or disinfected by the device; subjecting the defined surrogate protein having the predetermined sequence to sterilization, deimmunization, or disinfection; and determining the effectiveness of the sterilization, deimmunization, and/or disinfection by determining if the defined surrogate protein having the predetermined sequence has been destroyed, wherein the sterilization, deimmunization, and/or disinfection occurs when the defined surrogate protein has been fragmented to yield a negative result in a protein analysis procedure.
 2. The method of claim 1 in which the defined surrogate protein is proteins critical for stability, growth and/or infectious capacity of infectious agents.
 3. The method of claim 1 in which the defined surrogate protein is a protein critical for stability, growth and/or infectious capacity of surrogate organisms of infectious agents.
 4. The method of claim 2 in which the infectious agent includes one or more of: an infectious protein, an infectious spore forming bacteria, an infectious vegetative bacteria, an infections fungus, and an infectious virus.
 5. The method of claim 1 in which the defined surrogate protein includes pathogenic proteins, proteins critical for the growth of infectious agents, and Immunogenic proteins.
 6. The method of claim 1 in which the protein analysis procedure includes one or more of: a Western Blot analysis, a protein assay analysis, a magnetic separation analysis, a peptide analysis, a mass spectrometry analysis, and a gas chromatography analysis.
 7. The method of claim 1 in which the protein analysis procedure includes fluorescence analysis of proteins covalently crosslinked on a solid surface.
 8. The method of claim 1 in which the protein analysis procedure includes fluorescence analysis of proteins covalently crosslinked on magnetic beads.
 9. The method of claim 1 in which the defined surrogate protein having the predetermined sequence is disposed on a surface.
 10. The method of claim 1 in which the defined surrogate protein having the predetermined sequence is disposed on a test strip.
 11. The method of claim 1 in which the defined surrogate protein having the predetermined sequence is disposed in or on a vessel.
 12. The method of claim 1 in which the surrogate protein having the predetermined sequence is disposed on a tube.
 13. The method of claim 1 in which the surrogate protein having the predetermined sequence is disposed on a holder.
 14. The method of claim 13 in which the holder is disposed to receive a flow of a sterilization agent, a deimmunization agent or a disinfection agent.
 15. A method for determining effective sterilization, deimmunization, and/or disinfection of equipment and/or supplies by a device, the method comprising: providing a defined surrogate protein having a predetermined sequence defined by the sequence: (SEQ ID NO. 2)         10         20         30         40 MTLEKTYYEV TMEECTHQLH RDLPPTRLWG YNGLFPGPTI         50 EVKRNENVYV KWMNNLPSTH FLPIDHTIHH SDSQHEEPEV KTVVHLHGGV TPDDSDGYPE AWFSKDFEQT GPYFKREVYH YPNQQRGAIL WYHDHAMALT RLNVYAGLVG AYIIHDPKEK RLKHHHHHH

representative of an infectious agent potentially contaminating the equipment and/or the supplies to be sterilized, deimmunized, and/or disinfected by the device; subjecting the defined surrogate protein having the predetermined sequence to sterilization, deimmunization, or disinfection; and determining the effectiveness of the sterilization, deimmunization, and/or disinfection by determining if the defined surrogate protein having the predetermined sequence has been destroyed, wherein the sterilization, deimmunization, and/or disinfection occurs when the defined surrogate protein has been fragmented to yield a negative result in a protein analysis procedure. 